3-heteroarylidenyl-2-azaindolinones active as protein tyrosine kinase inhibitors

ABSTRACT

The invention relates to 3-heteroarylidenyl-2-azaindolinone compounds of the formula:                    
     wherein, 
     A is selected from the group consisting of nitrogen, oxygen and sulfur; 
     only one of B, D, E, F or G is nitrogen; 
     Z is selected from the group consisting of oxygen, sulfur and NR 11 ; or 
     physiologically acceptable salts thereof; 
     and wherein the groups R 1 -R 7  are defined herein. The 3-heteroarylidenyl-2-azaindolinone compounds of the preferred embodiments of the present invention have improved hydrosolubility and are expected to modulate the activity of protein tyrosine kinases. The 3-heteroarylidenyl-2-azaindolinone compounds of the preferred embodiments of the present invention should be useful in the prevention and treatment of protein tyrosine kinase related cellular disorders such as cancer.

RELATED APPLICATIONS

This Application is a Divisional of application Ser. No. 09/100,854,filed on Jun. 19, 1998 Now U.S. Pat. No. 6,313,158, which claimspriority from Provisional Application Serial No. 60/050,412, filed Jun.20, 1997, and Provisional Application Serial No. 60/059,336, filed Sep.19, 1997, all of which are incorporated by reference as if fully setforth herein.

INTRODUCTION

The present invention relates generally to organic chemistry,biochemistry, pharmacology and medicine. More particularly, it relatesto novel heterocyclic compounds, and their physiologically acceptablesalts and prodrugs, which modulate the activity of protein tyrosinekinases (“PTKs”) and, therefore, are expected to exhibit a salutaryeffect against disorders related to abnormal PTK activity.

BACKGROUND OF THE INVENTION

The following is offered as background information only and is notadmitted to be prior art to the present invention.

Growth factor receptors are cell-surface proteins. When bound by agrowth factor ligand, growth factor receptors are converted to an activeform which interacts with proteins on the inner surface of a cellmembrane. This leads to phosphorylation on tyrosine residues of thereceptor and other proteins and to the formation inside the cell ofcomplexes with a variety of cytoplasmic signaling molecules that, inturn, affect numerous cellular responses such as cell division(proliferation), cell differentiation, cell growth, expression ofmetabolic effects to the extracellular microenvironment, etc. For a morecomplete discussion, see Schlessinger and Ullrich, Neuron, 9:303-391(1992) which is incorporated by reference, including any drawings, as iffully set forth herein.

Growth factor receptors with PTK activity, known as receptor tyrosinekinases (“RTKs”), comprise a large family of transmembrane receptorswith diverse biological activity. At present, at least nineteen (19)distinct subfamilies of RTKs have been identified. An example of theseis the subfamily designated the “HER” RTKs, which include EGFR(epithelial growth factor receptor), HER2, HER3 and HER4. These RTKsconsist of an extracellular glycosylated ligand binding domain, atransmembrane domain and an intracellular cytoplasmic catalytic domainthat can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and the insulin receptor relatedreceptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II toform a heterotetramer of two entirely extracellular glycosylated αsubunits and two β subunits which cross the cell membrane and whichcontain the tyrosine kinase domain.

A third RTK subfamily is referred to as the platelet derived growthfactor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ, CSFIR,c-kit and c-fms. These receptors consist of glycosylated extracellulardomains composed of variable numbers of immunoglobin-like loops and anintracellular domain wherein the tyrosine kinase domains is interruptedby unrelated amino acid sequences.

Another group which, because of its similarity to the PDGFR subfamily,is sometimes subsumed in the later group is the fetus liver kinase(“flk”) receptor subfamily. This group is believed to be made of up ofkinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R,flk-4 and fms-like tyrosine kinase 1 (flt-1).

One further member of the tyrosine kinase growth factor receptor familyis the group known as the fibroblast growth factor (“FGF”)receptors.This group consists of four receptors, FGFR1-4, and seven ligands,FGF1-7. While not yet well defined, it appears that the receptorsconsist of a glycosylated extracellular domain containing a variablenumber of immunoglobin-like loops and an intracellular domain in whichthe PTK sequence is interrupted by regions of unrelated amino acidsequences.

A more complete listing of the known RTK subfamilies is described inPlowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases”. This latter designation, abbreviated “CTK”, will beused in this disclosure. CTKs do not contain extracellular andtransmembrane domains. At present, over 24 CTKs in 11 subfamilies (Src,Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have beenidentified. The Src subfamily appear so far to be the largest group ofCTKs and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For amore detailed discussion of CTKs, see Bolen, Oncogene, 8:2025-2031(1993), which is incorporated by reference, including any drawings, asif fully set forth herein.

Both RTKs and CTKs have been implicated in a host of pathogenicconditions including, significantly, cancer. Others include, withoutlimitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis,angiogenesis and a variety of renal disorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PTK regulated. That is, it has beensuggested that malignant cell growth results from a breakdown in themechanisms that control cell division and/or differentiation. It hasbeen shown that the protein products of a number of proto-oncogenes areinvolved in the signal transduction pathways that regulate cell growthand differentiation. These protein products of proto-oncogenes includethe extracellular growth factors, transmembrane growth factor PTKreceptors (RTKs) and cytoplasmic PTKs (CTKs), discussed above.

In view of the apparent link between PTK-related cellular activities anda number of human disorders, it is no surprise that a great deal ofeffort is being expended in an attempt to identify ways to modulate PTKactivity. Some of these have involved biomimetic approaches using largemolecules patterned on those involved in the actual cellular processes;e.g., mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors andantibodies (App. No. WO 94/10202, Kendall and Thomas, Proc. Nat'l Acad.Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844 (1993)); RNAligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano, et al.,Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62(1992); Wright, et al., J. Cellular Phys., 152:448-57)) and tyrosinekinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808;U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res.,35:2268 (1994)).

More recently, attempts have been made to identify small molecules whichact as PTK inhibitors. For example, bis-monocylic, bicyclic andheterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindolederivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones(U.S. Pat. No. 5,330,992) have been described as tyrosine kinaseinhibitors. Styryl compounds (U.S. Pat. No. 5,217,999),styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606),quinazoline derivatives (EP App. No. 0 566 266 A1), selenaindoles andselenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have allbeen described as PTK inhibitors with potential utility for thetreatment of cancer.

An area in need of improvement with regard to PTK-active compounds istheir bioavailability in vivo. It is not uncommon for a molecule toexhibit good PTK modulating activity in vitro, where it can be placed inimmediate proximity to the PTK of interest, but to have substantiallyless, sometimes no, activity in vivo. Without being bound to aparticular theory, applicants believe that this phenomenon may be due tothe fact that many molecules of interest as modulators of PTK activity,including indolinones, tend to be lipophilic. However, in many cases,the region where the PTKs reside and perform their function are aqueousin nature. Thus, the compounds might not be capable of reaching theactive site. Improving the hydrosolubility of indolinones could lead tocompounds with improved bioavailiabilty and thereby improved PTKmodulation in vivo.

SUMMARY OF THE INVENTION

Our efforts to identify small organic molecules which exhibit improvedhydrosolubility while maintaining their ability to modulate PTK activityand which, therefore, should be useful it the treatment and preventionof disorders driven by abnormal TK activity, has led us to the discoveryof a family of novel heterocyclic compounds which exhibit improvedhydrosolubility and still have the desired ability to modulate PTKactivity and which are the subject of this invention. Thus, the presentinvention relates generally to novel 3-heteroarylidenyl-2-indolinoneswhich have improved hydrosolubility and which modulate the activity ofboth receptor (RTK) and non-receptor (CTK) protein tyrosine kinases(PTKs). In addition, the present invention relates to the preparationand use of pharmacological compositions of the disclosed compounds andtheir physiologically acceptable salts and prodrugs in the treatment orprevention of PTK driven disorders such as, by way of example and notlimitation, cancer, diabetes, hepatic cirrhosis, atherosclerosis,angiogenesis and renal disease.

A “3-heteroarylidenyl-2-indolinone” refers to a chemical compound havinga “heteroaryl” group, as defined below, bonded to a carbon—carbon doublebond, the other end of which double bond is bonded to the ring carbon ofthe pyrrolidone ring of an indolin-2-one.

As used herein, “lipophilic” refers to molecules which have an affinityfor, or capability of dissolving in, lipids; i.e., non-water solubleoils, fats, sterols, triglycerides and the like.

The term “hydrosoluble” and “hydrosolubility” refer to molecules whichhave an affinity for, or capability of dissolving in, aqueous solutions;i.e., solutions consisting primarily of water.

The terms “indolin-2-one”, “2-indolinone” and “2-oxindole all refer to asix-member aryl fused through two adjacent ring carbons to a pyrrolidonering at the carbon adjacent to the ring nitrogen and to the carbon nextto that carbon.

When D, E, F or G is nitrogen the bicyclic ring is properly termed as“azaindolin-2-one.” However, for the purposes of this disclosure theterm indolin-2-one, 2-indolinone or 2-oxindole will be understood toincorporate the nitrogen-containing compounds as well.

A “pyrrolidone” ring has the structure:

As used herein, the term “heteroarylidenyl” group refers to the groupconsisting of the double bond and the heteroaryl group and R² bonded toit.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or physiologically acceptable salts orprodrugs thereof, with other chemical components, such asphysiologically acceptable carriers and excipients. The purpose of apharmacological composition is to facilitate administration of acompound to an organism.

A “prodrug” refers to an agent which is converted into the parent drugin vivo. Prodrugs are often useful because, in some situations, they maybe easier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmacologicalcompositions over the parent drug. An example, without limitation, of aprodrug would be a compound of the present invention wherein it isadministered as an ester (the “prodrug”) to facilitate transmittalacross a cell membrane where water solubility is not beneficial, butthen it is metabolically hydrolyzed to the carboxylic acid once insidethe cell where water solubility is beneficial.

As used herein, an “ester” is a carboxy group, as defined herein,wherein “R” is any of the listed groups other than hydrogen.

As used herein, a “physiologically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

An “excipient” refers to an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

1. The Compounds

A. General Structural Features.

In one aspect, this invention relates to a3-hetero-arylidenyl-2-indolinone compound which is substituted with oneor more groups which have an affinity for combining with water and whichtherefore improve the hydrosolubility of the3-heteroarylidenyl-2-indolinone.

By “affinity for combining with water” is meant that the group, in thepresence of water, interacts electronically with water moleculesthrough, but not limited to, such mechanisms as ionization, hydrogenbonding and hydration to form complexes with the water molecules whichcan improve the solubility of the entire molecule in water.

By “improve the hydrosolubility” is meant that the compound substitutedwith one or more of the indicated groups is more soluble in water thanthe same molecule without the indicated groups.

In another aspect, the present invention relates to3-heteroarylidenyl-2-indolinones having the chemical structure shown inFormula 1:

A is selected from the group consisting of nitrogen, oxygen and sulfurand it is understood that when A is oxygen or sulfur, R³ does not exist.

B, D, E, F and G are independently selected from the group consisting ofcarbon and nitrogen and it is understood that when B, D, E, F or G isnitrogen, R⁴, R⁵, R⁶ or R⁷, respectively, do not exist.

Z is selected from the group consisting of oxygen, sulfur and NR¹¹.

R¹¹ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl, C-carboxy,O-carboxy, C-amido, guanyl, sulfonyl and trihalomethanesulfonyl.

R¹ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, hydroxy, alkoxy, C-carboxy, C-amido,trihalomethanecarbonyl, trihalomethanesulfonyl and sulfonyl.

R² is selected from the group consisting of hydrogen, alkyl, cycloalkyl,aryl and halogen.

When A is nitrogen, R³ is selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl,C-carboxy, O-carboxy, C-amido, guanyl, sulfonyl andtrihalomethanesulfonyl.

One or two of R⁴, R⁵, R⁶ and R⁷ are independently selected from thegroup consisting of —NR⁸R⁹, —J(CH₂)_(m)—NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q,—N═CNR⁸R⁹ and —NHR¹⁰.

J is selected from the group consisting of oxygen, nitrogen and sulfur.

The subscript m can be 0, 1, 2 or 3.

Y is selected from the group consisting of —NH and oxygen.

Q is selected from the group consisting of hydroxy, alkoxy, aryloxy,amino, N-hydroxylamino, O-carboxy, NR⁸R⁹ and N-peptidyl. R⁸ and R⁹ areindependently selected from the group consisting of hydrogen, alkyl,C-carboxy, C-peptidyl and, combined, a five-member or 6-memberheteroalicyclic group containing at least one nitrogen.

R¹⁰ is a polyhydroxyalkyl group.

The remaining groups are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, S-sulfonamido,N-Sulfonamido, trihalomethanesulfonyl, carbonyl, C-carboxy, O-carboxy,C-amido, N-amido, cyano, nitro, halo, O-thiocarbamyl, N-thiocarbamyl,guanyl and phosphonyl; and,

R⁴ and R⁵ may combine to form a six-member cycloalkyl, heteroaryl orheteroalicyclic ring.

As used herein, the term “alkyl” refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms (whenever anumerical range; e.g. “1-20”, is stated herein, it means that the group,in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms,3 carbon atoms, etc. up to and including 20 carbon atoms). Morepreferably, it is a medium size alkyl having 1 to 10 carbon atoms. Mostpreferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkylgroup may be substituted or unsubstituted. When substituted, thesubstituent group(s) is preferably one or more individually selectedfrom cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, sulfonamido,trihalomethane-sulfonamido, silyl, guanyl, guanidino, ureido, amino andNR¹²R¹³, wherein R¹² and R¹³ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, sulfonyl,trihalomethysulfonyl and, combined, a five- or six-memberheteroalicyclic ring.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more individually selectedfrom alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, nitro, guanyl, ureido, guanidino,amino and NR¹²R¹³, with R¹² and R¹³ being as defined herein.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon—carbondouble bond.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon—carbontriple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more selectedfrom halo, trihalomethyl, alkyl, alkenyl, alkynyl, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, amino andNR¹²R¹³ wherein R¹² and R¹³ are previously defined herein.

As used herein, a “heteroaryl” group refers to a monocyclic or fusedring (i.e., rings which share an adjacent pair of atoms) group having inthe ring(s) one or more atoms selected from the group consisting ofnitrogen, oxygen and sulfur and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofheteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline,purine and carbazole. The heteroaryl group may be substituted orunsubstituted. When substituted, the substituted group(s) is preferablyone or more selected from alkyl, cycloalkyl, halo, trihalomethyl,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, O-carboxy,sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,trihalomethanesulfonamido, amino and NR¹²R¹³ where R¹² and R¹³ arepreviously defined herein.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms selected from the groupconsisting of nitrogen, oxygen and sulfur. The rings may also have oneor more double bonds. However, the rings do not have a completelyconjugated pi-electron system. The heteroalicyclic ring may besubstituted or unsubstituted. When substituted, the substituted group(s)is preferably one or more selected from alkyl, cycloaklyl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl,sulfonyl, S-sulfonamido, N-sulfonamido, C-amido, N-amido, ureido,guanyl, guanidino, amino and N¹²R¹³ where R¹² and R¹³ are previouslydefined herein.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “thiohydroxy” group refers to an —SH group.

A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “carbonyl” group refers to a —C(═O)—R″ group, where R″ is selectedfrom the group consisting of hydrogen, alkyl, cycloalkyl, aryl,heteroaryl (bonded through a ring carbon) and heteroalicyclic (bondedthrough a ring carbon), as defined herein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as definedherein.

A “trihalomethanecarbonyl” group refers to a X₃CC (═O)— group with X asdefined herein.

A “C-carboxy” group refers to a —C(═O)O—R″ groups, with R″ as definedherein.”An “O-carboxy” group refers to a R″C(═O)O— group, with R″ asdefined herein.

A “carboxylic acid” group refers to a C-carboxy group in which R″ ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groups with X asdefined above.

A “trihalomethanesulfonamido” group refers to a X₃CS (═O)₂NR¹²— groupwith X and R¹² as defined herein.

A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ as definedherein.

A “sulfonyl” group refers to a —S(═O)₂R″ group, with R″ as definedherein.

An “S-sulfonamido” group refers to a —S(═O)₂NR¹²R¹³, with R¹² and R¹³ asdefined herein.

An “N-Sulfonamido” group refers to a R¹²S(═O)₂NR¹³— group, with R¹² andR¹³ as defined herein.

An “O-carbamyl” group refers to a —OC(═O)NR¹²R¹³ group with R¹² and R¹³as defined herein.

An “N-carbamyl” group refers to a R¹²OC(═O)NR¹³— group, with R¹² and R¹³as defined herein.

An “O-thiocarbamyl” group refers to a —OC(═S)NR¹²R¹³ group with R¹² andR¹³ as defined herein.

An “N-thiocarbamyl” group refers to a R¹²OC(═S)NR¹³— group, with R¹² andR¹³ as defined herein.

An “amino” group refers to an —NR¹²R¹³ group, with R¹² and R¹³ asdefined herein.

A “C-amido” group refers to a —C(═O)NR¹²R¹³ group with R¹² and R¹³ asdefined herein.

An “N-amido” group refers to a R¹²C(═O)NR¹³— group, with R¹² and R¹³ asdefined herein.

A “quaternary ammonium” group refers to a —⁺NHR¹²R¹³ group wherein R¹²and R¹¹ are independently selected from the group consisting of alkyl,cycloalkyl, aryl, and heteroaryl.

A “ureido” group refers to a —NR¹²C(═O)NR¹³R¹⁴ group, with R¹² and R¹³as defined herein and R¹⁴ defined the same as R¹² and R¹³.

A “guanidino” group refers to a —R¹²NC(═N)NR¹³R¹⁴ group, with R¹², R¹³and R¹⁴ as defined herein.

A “guanyl” group refers to a R¹²R¹³NC(═N)— group, with R¹² and R¹³ asdefined herein.

A “nitro” group refers to a —NO₂ group.

A “cyano” group refers to a —C≡N group.

A “silyl” group refers to a —Si(R″)₃ group, with R″ as defined herein.

An “N-hydroxylamino” group refers to a —NHOR″ group, with R″ as definedherein.

A “polyhydroxyalkyl” group refers to a 1 to 8 carbon, preferably a 1 to4 carbon straight chain alkyl group substituted with 2 or more,preferrably 3 or more, hydroxyl groups.

A “peptidyl” group generally refers to a group formed by the interactionbetween the amino groups and the carboxy groups of amino acids. Apeptidyl group has the general formula:

wherein the Rs may be the same or different. The amino acid on the lefthand side of the above formula is referred to as the N-terminal aminoacid residue and the amino acid on the right hand side is referred to asthe C-terminal amino acid residue.

An “N-peptidyl” group refers to a peptidyl group which is bonded throughthe N-terminal amino acid to a non-amino acid molecule.

A “C-peptidyl” group refers to a peptidyl group which is bonded throughthe C-terminal amino acid to a non-amino acid molecule.

B. Preferred Structural Features.

Preferred structural features of this invention are those in which:

R¹ is selected from the group consisting of hydrogen, alkyl, hydroxy,alkoxy and C-carboxy;

Z is selected from the group consisting of sulfur and oxygen;

R² is selected from the group consisting of hydrogen and alkyl;

A and B are nitrogen;

R³ is selected from the group consisting of hydrogen and alkyl; and,

R⁴ and R⁵ are independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, C-carboxy, alkoxy, cyano, andC-carboxy.

Additional preferred structures of the present invention are those inwhich:

A is sulfur; and,

R⁴ and R⁵, combined, form a six-member cycloalkyl, heteroaryl orheteroalicyclic ring.

Still further preferred embodiments of the present invention are thosein which:

A is nitrogen;

B is carbon;

R¹ is hydrogen; and,

R⁴ and R⁵ are lower alkyl.

The chemical formulae referred to herein may exhibit the phenomena oftautomerism and structural isomerism. For example, the compoundsdescribed herein may adopt a cis or trans configuration about the doublebond connecting the indolin-2-one moiety to the heteroaryl moiety orthey may be a mixture of cis and trans. This invention encompasses anytautomeric or structural isomeric form and mixtures thereof whichpossess the ability to modulate RTK and/or CTK activity and is notlimited to any one tautomeric or structural isomeric form.

As used herein, the term “cis” refers to the structural configurationwherein the heteroaryl group is on the same side of the double bondconnecting it to the indolin-2-one ring as the 2-oxygen group of theindolin-2-one.

As used herein, the term “trans” refers to the structural configurationwherein the heteroaryl group is on the opposite side of the double bondconnecting it to the indolin-2-one ring as the 2-oxygen group of theindolin-2-one.

2. The Biochemistry

In yet another embodiment, this invention relates to a method for themodulation of the catalytic activity of PTKs comprising administering acompound of this invention or a physiologically acceptable salt or aprodrug thereof to a PTK.

By “PTK” is meant both RTKs and CTKs; i.e., the modulation of both RTKsignal transduction and CTK signal transduction is contemplated by thisinvention.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

As used herein, the term “modulation” or “modulating” refers to thealteration of the catalytic activity of RTKs and/or CTKs. In particular,modulating refers to the activation of the catalytic activity of RTKsand/or CTKs, more preferably the activation or inhibition of thecatalytic activity of RTKs and/or CTKs, depending on the concentrationof the compound administered or, more preferably still, the inhibitionof the catalytic activity of RTKs and/or CTKs.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect ofRTKs and/or CTKS.

The term “administering” as used herein refers to a method for bringinga compound of this invention and a target PK together in such a mannerthat the compound can affect the catalytic activity of the PK eitherdirectly; i.e., by interacting with the kinase itself, or indirectly;i.e. by interacting with another molecule on which the catalyticactivity of the kinase is dependent. As used herein, administration canbe accomplished either in vitro, i.e. in a test tube, or in vivo, i.e.in cells or tissues of a living organism. Thus, the TK mediateddisorders which are the object of this invention can be studied,prevented or treated by the methods set forth herein whether the cellsor tissues of the organism exist within the organism or outside theorganism. Cells existing outside the organism can be maintained or grownin cell culture dishes. In this context, the ability of a particularcompound to affect a PTK related disorder can be determined; i.e., theIC50 of the compound, defined below, before the use of the compounds inmore complex living organisms is attempted. For cells outside theorganism, multiple methods exist, and are well-known to those skilled inthe art, to administer compounds including, but not limited to, directcell microinjection and numerous transmembrane carrier techniques. Forcells harbored within a living organism, myriad methods also exist, andare likewise well-known to those skilled in the art, to administercompounds including, but not limited to, oral, parenteral, dermal andaerosol applications.

RTK mediated signal transduction is initiated by extracellularinteraction with a specific growth factor (ligand), followed by receptordimerization, transient stimulation of the intrinsic protein tyrosinekinase activity and phosphorylation. Binding sites are thereby createdfor intracellular signal transduction molecules and lead to theformation of complexes with a spectrum of cytoplasmic signalingmolecules that facilitate the appropriate cellular response (e.g., celldivision, metabolic effects to the extracellular microenvironment). See,Schlessinger and Ullrich, 1992, Neuron 9:303-391.

It has been shown that tyrosine phosphorylation sites in growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423;Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785); Songyang et al.,1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates which have a catalytic domain; and (2) substrates which lacksuch domain but serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767-778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

PTK signal transduction results in, among other responses, cellproliferation, differentiation, growth and metabolism. Abnormal cellproliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis,arthritis and diabetic retinopathy (or other disorders related touncontrolled angiogenesis and/or vasculogenesis).

A precise understanding of the mechanism by which the compounds of thisinvention inhibit PTKs is not required in order to practice the presentinvention. However, while not being bound to any particular mechanism ortheory, it is believed that the compounds interact with the amino acidsof the catalytic region of PTKs. PTKs typically possess a bi-lobatestructure wherein ATP appears to bind in the cleft between the two lobesin a region where the amino acids are conserved among PTKs. Inhibitorsof PTKs are believed to bind by non-covalent interactions such ashydrogen bonding, van der Waals forces and ionic interactions in thesame general region where the aforesaid ATP binds to the PTKs. Morespecifically, it is thought that the indolinone component of thecompounds of this invention binds in the general space normally occupiedby the adenine ring of ATP. Specificity of a particular molecule for aparticular PTK could arise as the result of additional interactionsbetween the various substituents on the indolinone core with amino aciddomains specific to particular PTKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PTKs.The ability to select those compounds active at different ATP (or othernucleotide) binding sites makes the compounds useful for targeting anyprotein with such a site; i.e., not only PTKs but serine/threoninekinases and protein phosphatases as well. Thus, the compounds disclosedherein have utility for in vitro assays on such proteins and for in vivotherapeutic effects through such proteins.

Thus, in another aspect, this invention relates to a method for treatingor preventing a PTK related disorder by administering a therapeuticallyeffective amount of a compound of this invention or a salt or a prodrugthereof to an organism.

As used herein, “PTK related disorder,” “PTK driven disorder,” and“abnormal PTK activity” all refer to a disorder characterized byinappropriate or over-activity of PTKs, which can be either RTKs orCTKs. Inappropriate activity refers to either: (1) PTK expression incells which normally do not express PTKs; (2) increased PTK expressionleading to unwanted cell proliferation, differentiation and/or growth;or, (3) decreased PTK expression leading to unwanted reductions in cellproliferation, differentiation and/or growth. Overactivity of PTKsrefers to either amplification of the gene encoding a particular PTK orproduction of a level of PTK activity which can correlate with a cellproliferation, differentiation and/or growth disorder (that is, as thelevel of the PTK increases, the severity of one or more of the symptomsof the cellular disorder increases).

As used herein, the terms “prevent”, “preventing” and “prevention” referto a method for barring an organism from the first place acquiring anPTK mediated cellular disorder.

As used herein, the terms “treat”, “treating” and “treatment” refer to amethod of alleviating or abrogating the PTK mediated cellular disorderand/or its attendant symptoms. With regard particularly to cancer, theseterms simply mean that the life expectancy or an individual affectedwith a cancer will be increased or that one or more of the symptoms ofthe disease will be reduced.

As used herein, the term “cancer” refers to various types of malignantneoplasms, most of which can invade surrounding tissues, and maymetastasize to different sites, as defined by Stedman's MedicalDictionary 25th edition (Hensyl ed. 1990). Examples of cancers which maybe treated by the present invention include, but are not limited to,brain, ovarian, colon, prostate, kidney, bladder, breast, lung, oral andskin cancers which exhibit inappropriate PTK activity. These types ofcancers can be further characterized. For example, brain cancers includeglioblastoma multiforme, anaplastic astrocytoma, astrocytoma,ependymoma, oligodendroglioma, medulloblastoma, meningioma, sarcoma,hemangioblastoma, and pineal parenchymal. Skin cancers include melanomaand Kaposi's sarcoma.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukariotic cell or as complex as a mammal, including a human being.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor; (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis; (3) inhibiting to some extent(that is slowing to some extent, preferably stopping) tumor growth;and/or, (4) relieving to some extent (or preferably eliminating) one ormore symptoms associated with the cancer.

This invention is therefore directed to compounds which modulate PTKsignal transduction by affecting the enzymatic activity of the RTKsand/or CTKs and thereby interfering with the signal transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate the RTK and/or CTK mediated signal transductionpathways as a therapeutic approach to cure many kinds of solid tumors,including but not limited to carcinoma, sarcoma, erythroblastoma,glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.Indications may include, but are not limited to brain cancers, bladdercancers, ovarian cancers, gastric cancers, pancreas cancers, coloncancers, blood cancers, lung cancers, bone cancers and leukemias.

Further examples, without limitation, of the types of disorders relatedto unregulated PTK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders which may be prevented, treated or furtherstudied by the present invention include cancers, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to angiogenic andvasculogenic disorders generally resulting in abnormal proliferation ofblood vessels. The formation and spreading of blood vessels, orvasculogenesis and angiogenesis, respectively, play important roles in avariety of physiological processes such as embryonic development, corpusluteum formation, wound healing and organ regeneration. They also play apivotal role in cancer development. Other examples of blood vesselproliferation disorders include arthritis, where new capillary bloodvessels invade the joint and destroy cartilage, and ocular diseases,like diabetic retinopathy, where new capillaries in the retina invadethe vitreous, bleed and cause blindness. Conversely, disorders relatedto the shrinkage, contraction or closing of blood vessels, such asrestenosis, are also implicated.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. Hepatic cirrhosis cancause diseases such as cirrhosis of the liver. An increasedextracellular matrix resulting in a hepatic scar can also be caused byviral infection such as hepatitis. Lipocytes appear to play a major rolein hepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes, transplant rejection, andglomerulopathies. For instance, PDGFR has been implicated in themaintenance of mesangial cell proliferation. Floege et al., 1993, KidneyInternational 43:47S-54S.

As noted previously, PTKs have been associated with cell proliferativedisorders. For example, some members of the RTK family have beenassociated with the development of cancer. Some of these receptors, likeEGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992,APMIS 100:713-719); HER2/neu (Slamon et al., 1989, Science 244:707-712)and PDGFR (Kumabe et al., 1992, Oncogene, 7:627-633) are over-expressedin many tumors and/or are persistently activated by autocrine loops. Infact, in the most common and severe cancers these receptorover-expressions and autocrine loops have been demonstrated (Akbasak andSuner-Akbasak et al., 1992, J. Neurol. Sci., 111:119-133; Dickson etal., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin.Invest. 90:1352-1360); (Lee and Donoghue, 1992, J. Cell. Biol.,118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al.,supra). For example, the EGFR receptor has been associated with squamouscell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lungcancer and bladder cancer. HER2 has been associated with breast,ovarian, gastric, lung, pancreas and bladder cancer. PDGFR has beenassociated with glioblastoma, lung, ovarian, melanoma and prostate. TheRTK c-met has been generally associated with hepatocarcinogenesis andthus hepatocellular carcinoma. Additionally, c-met has been linked tomalignant tumor formation. More specifically, the RTK c-met has beenassociated with, among other cancers, colorectal, thyroid, pancreaticand gastric carcinoma, leukemia and lymphoma. Additionally,over-expression of the c-met gene has been detected in patients withHodgkins disease, Burkitts disease, and the lymphoma cell line.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while being integrally involved in the normal growthand differentiation of the nervous system, appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of the IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cellsof the bone marrow) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series ofrecent publications, Baserga even suggests that IGF-IR plays a centralrole in the mechanisms of transformation and, as such, could be apreferred target for therapeutic interventions for a broad spectrum ofhuman malignancies. Baserga, 1995, Cancer Res., 55:249-252; Baserga,1994, Cell 79:927-930; Coppola et al., 1994, Mol. Cell. Biol.,14:4588-4595.

The association between abnormal RTK activity and disease are notrestricted to cancer, however. For example, RTKs have been associatedwith metabolic diseases like psoriasis, diabetes mellitus, woundhealing, inflammation, and neurodegenerative diseases. For example, EGFRhas been indicated in corneal and dermal wound healing. Defects in theInsulin-R and IGF-1R are indicated in type-II diabetes mellitus. A morecomplete correlation between specific RTKs and their therapeuticindications is set forth in Plowman et al., 1994, DN&P 7:334-339.

As noted previously, not only RTKs but CTKs as well including, but notlimited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk(reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involved inthe proliferative and metabolic signal transduction pathway and thuswere expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been demonstrated as an oncoprotein(pp60^(v-src)) in chicken. Moreover, its cellular homolog, theproto-oncogene pp60^(c-src) transmits oncogenic signals of manyreceptors. For example, over-expression of EGFR or HER2/neu in tumorsleads to the constitutive activation of pp60^(c-src), which ischaracteristic for the malignant cell but absent from the normal cell.On the other hand, mice deficient in the expression of c-src exhibit anosteopetrotic phenotype, indicating a key participation of c-src inosteoclast function and a possible involvement in related disorders.Similarly, Zap70 is implicated in T-cell signaling.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

3. Pharmacologcal Compositions and Therapeutic Applications

A compound of the present invention, or its physiologically acceptablesalt or prodrug, can be administered to a human patient per se, or inpharmacological compositions where it is mixed with suitable carriers orexcipient(s). Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

A. Routes Of Administration.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal or intranasal injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

B. Composition/Formulation.

Pharmacological compositions of the compounds and the physiologicallyacceptable salts and prodrugs thereof are preferred embodiments of thisinvention. Pharmacological compositions of the present invention may bemanufactured by processes well known in the art; e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmacological compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmacological preparations fororal use can be made with the use of a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragée cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmacological compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmacological compositions for parenteral administration includeaqueous solutions of the active compounds in water soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The pharmacological compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PTK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate,succinate, etc. formed by the reaction of an amino group with theappropriate acid. Salts in which the compound forms the negativelycharged species include, without limitation, the sodium, potassium,calcium and magnesium salts formed by the reaction of a carboxylic acidgroup in the molecule with the appropriate base (e.g. sodium hydroxide(NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca (OH₂), etc.).

C. Dosage.

Pharmacological compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve its intended purpose.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. For example, a dose can be formulated in animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in cell culture (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of the PTK activity).Such information can be used to more accurately determine useful dosesin humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g.,Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thekinase modulating effects, or minimal effective concentration (MEC). TheMEC will vary for each compound but can be estimated from in vitro data;e.g., the concentration necessary to achieve 50-90% inhibition of thekinase using the assays described herein. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. However, HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

D. Packaging.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor human or veterinary administration. Such notice, for example, may beof the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

4. Synthesis

The compounds of this invention, as well as the precursor indolin-2-onesand aldehydes, may be readily synthesized using techniques well known inthe chemical arts. It will be appreciated by those skilled in the artthat other synthetic pathways for forming the compounds of the inventionare available and that the following is offered by way of example andnot limitation.

A. General Synthetic Procedure.

The following general methodology may be employed to prepare thecompounds of this invention:

The appropriately substituted indolin-2-one (1 equiv.), theappropriately substituted aldehyde (1.2 equiv.) and piperidine (0.1equiv.) are mixed with ethanol (1-2 ml/mmol 2-indolinone) and themixture is then heated at 90° C. for 3 to 5 hours After cooling, theprecipitate is filtered, washed with cold ethanol and dried to yield thetarget compound.

B. 2-oxindoles

The following examples show representative syntheses of several of the2-oxindole precursors to the compounds of this invention. These2-oxindoles, as well as the others claimed, will form the claimedcompounds by reaction with an appropriately substituted aldehyde underthe conditions described above. It is to be understood that thefollowing syntheses are provided by way of example only and are not tobe construed as limiting as to synthetic procedure or as to thecompounds described.

5-Amino-2-oxindole

5-Nitro-2-oxindole (6.3 g) was hydrogenated in methanol over 10%palladium on carbon to give 3.0 g (60% yield) of the title compound as awhite solid.

5-Bromo-2-oxindole

2-Oxindole (1.3 g) in 20 mL acetonitrile was cooled to −10° C. and 2.0 gN-bromosuccinimide was slowly added with stirring. The reaction wasstirred for 1 hour at −10° C. and 2 hours at 0° C. The precipitate wascollected, washed with water and dried to give 1.9 g (90% yield) of thetitle compound.

4 -Methy-2-oxindole

Diethyl oxalate (30 mL) in 20 mL of dry ether was added with stirring to19 g of potassium ethoxide suspended in 50 mL of dry ether. The mixturewas cooled in an ice bath and 20 mL of 3-nitro-o-xylene in 20 mL of dryether was slowly added. The thick dark red mixture was heated to refluxfor 0.5 hr, concentrated to a dark red solid, and treated with 10%sodium hydroxide until almost all of the solid dissolved. The dark redmixture was treated with 30% hydrogen peroxide until the red colorchanged to yellow. The mixture was treated alternately with 10% sodiumhydroxide and 30% hydrogen peroxide until the dark red color was nolonger present. The solid was filtered off and the filtrate acidifiedwith 6N hydrochloric acid. The resulting precipitate was collected byvacuum filtration, washed with water, and dried under vacuum to give 9.8g (45% yield) of 2-methyl-6-nitrophenylacetic acid as an off-whitesolid. The solid was hydrogenated in methanol over 10% palladium oncarbon to give 9.04 g of the title compound as a white solid.

7-Bromo-5-chloro-2-oxindole

5-Chloro-2-oxindole (16.8 g) and 19.6 g of N-bromosuccinimide weresuspended in 140 mL of acetonitrile and refluxed for 3 hours. Thin layerchromatography (silica, ethyl acetate) at 2 hours of reflux showed5-chloro-2-oxindole or N-bromosuccinimide (Rf 0.8), product (Rf 0.85)and a second product (Rf 0.9) whose proportions did not change afteranother hour of reflux. The mixture was cooled to 10° C., theprecipitate was collected by vacuum filtration, washed with 25 mL ofethanol and sucked dry for 20 minutes in the funnel to give 14.1 g ofwet product (56% yield). The solid was suspended in 200 mL of denaturedethanol and slurry-washed by stirring and refluxing for 10 minutes. Themixture was cooled in an ice bath to 10° C. The solid product wascollected by vacuum filtration, washed with 25 mL of ethanol and driedunder vacuum at 40° C. to give 12.7 g (51% yield) of7-bromo-5-chloro-2-oxindole.

5-Fluoro-2-oxindole

5-Fluoroisatin (8.2 g) was dissolved in 50 mL of hydrazine hydrate andrefluxed for 1.0 hr. The reaction mixtures were then poured in icewater. The precipitate was then filtered, washed with water and driedunder vacuum oven afford the title compound.

5 -Nitro-2 -oxindole

2-Oxindole (6.5 g) was dissolved in 25 mL concentrated sulfuric acid andthe mixture maintained at −10 to −15° C. while 2.1 mL of fuming nitricacid was added dropwise. After the addition of the nitric acid thereaction mixture was stirred at 0° C. for 0.5 hr and poured intoice-water. The precipitate was collected by filtration, washed withwater and crystallized from 50% acetic acid. The crystalline product wasthen filtered, washed with water and dried under vacuum to give 6.3 g(70%) of 5-nitro-2-oxindole.

5-Iodo-2-oxindole

2-Oxindole (82.9 g) was suspended in 630 mL of acetic acid withmechanical stirring and the mixture cooled to 10° C. in an ice waterbath. Solid N-iodosuccinimide (175 g) was added in portions over 10minutes. After the addition was complete the mixture was stirred for 1.0hour at 10° C. The suspended solid which had always present became verythick at this time. The solid was collected by vacuum filtration, washedwith 100 mL of 50% acetic acid in water and then with 200 mL of waterand sucked dry for 20 minutes in the funnel. The product was dried undervacuum to give 93.5 g (36%) of 5-iodo-2-oxindole containing about 5%2-oxindole by proton NMR.

5-Methyl-2-oxindole

5-Methylisatin (15.0 g) and 60 mL of hydrazine hydrate were heated at140 to 160° C. for 4 hours. Thin layer chromatography (ethylacetate:hexane 1:2, silica gel) showed no starting material remaining.The reaction mixture was cooled to room temperature, poured into 300 mLof ice water and acidified to pH 2 with 6N hydrochloric acid. Afterstanding at room temperature for 2 days the precipitate was collected byvacuum filtration, washed with water and dried under vacuum to give 6.5g (47% yield) of 5-methyl-2-oxindole.

5-Bromo-4-methyloxindole and 5,7-Dibromo-4-methyloxindole

4-Methyl-2-oxindole (5 g) in 40 mL of acetonitrile was treated with 7.26g of N-bromosuccinimide and stirred at room temperature for 4 hours.Thin layer chromatography (ethyl acetate:hexane 1:2, silica gel) showeda mixture of 5-bromo (Rf 0.3) and 5,7-dibromo (Rf 0.5) products. Another7.26 g of N-bromosuccinimide was added and the mixture stirred for 4additional hours. The solid was collected by vacuum filtration, washedwith 20 mL of acetonitrile and dried to give a 1:1 mixture of mono anddibromo compounds. The filtrate was concentrated and chromatographed onsilica gel (ethyl acetate:hexane (1:2)) to give 1.67 g of5-bromo-4-methyl-2-oxindole as a beige solid. The remaining 1:1 mixtureof solids was recrystallized twice from glacial acetic acid to give 3.2g of 5,7-dibromo-4-methyl-2-oxindole as a light orange solid. Thefiltrates from this material were chromatographed as above to give 0.6 gof 5-bromo-4-methyl-2-oxindole and 0.5 g of5,7-dibromo-4-methyl-2-oxindole.

6-Fluoro-2-oxindole

Sodium hydride (2.6 g) and 14.5 g of dimethylmalonate was stirred andheated to 100° C. in 160 mL dimethylsulfoxide for 1.0 hour. The mixturewas cooled to room temperature, 7.95 g of 2,5-difluoronitrobenzene wereadded and mixture stirred for 30 minutes. The mixture was then heated to100° C. for 1.0 hour, cooled to room temperature and poured into 400 mLof saturated ammonium chloride solution. The mixture was extracted with200 mL of ethyl acetate and the organic layer washed with brine, driedover anhydrous sodium sulfate and concentrated under vacuum. The residuewas crystallized from methanol to give 24.4 g (80% yield) of dimethyl4-fluoro-2-nitrophenylmalonate as a white solid, Rf 0.2 on thin layerchromatography (ethyl acetate:hexane 1:6, silica gel). The filtrate wasconcentrated and chromatographed on a column of silica gel (ethylacetate:hexane 1:8) to give an additional 5.03 g of dimethyl4-fluoro-2-nitro-phenylmalonate, for a total of 29.5 g (96% yield).

Dimethyl 4-fluoro-2-nitrophenylmalonate (5.0 g) was refluxed in 20 mL of6N hydrochloric acid for 24 hours. The reaction was cooled and the whitesolid collected by vacuum filtration, washed with water and dried togive 3.3 g (87% yield) of 4-fluoro-2-nitrophenylacetic acid, Rf 0.6 onthin layer chromatography (ethyl acetate:hexane 1:2, silica gel).

4-Fluoro-2-nitrophenylacetatic acid (3.3 g) dissolved in 15 mL of aceticacid was hydrogenated over 0.45 g of 10% palladium on carbon at 60 psiH₂ for 2 hours. The catalyst was removed by filtration and washed with15 mL of methanol. The combined filtrates were concentrated and dilutedwith water. The precipitate was collected by vacuum filtration, washedwith water and dried to give 1.6 g (70% yield) of 6-fluoro-2-oxindole,Rf 0.24 on thin layer chromatography. The filtrate was concentrated togive a purple solid with an NNM spectrum similar to the first crop.Chromatography of the purple solid (ethyl acetate:hexane 1:2, silicagel) gave a second crop of 6-fluoro-2-oxindole as a white solid.

5-Aminosulfonyl-2-oxindole

To a 100 mL flask charged with 27 mL of chlorosulfonic acid was addedslowly 13.3 g of 2-oxindole. The reaction temperature was maintainedbelow 30° C. during the addition. After the addition, the reactionmixture was stirred at room temperature for 1.5 hr, heated to 68° C. forIhr, cooled, and poured into water. The precipitate was washed withwater and dried in a vacuum oven to give 11.0 g of5-chlorosulfonyl-2-oxindole (50% yield) which was used without furtherpurification.

5-Chlorosulfonyl-2-oxindole (2.1 g) was added to 10 mL of ammoniumhydroxide in 10 mL of ethanol and stirred at room temperature overnight.The mixture was concentrated and the solid collected by vacuumfiltration to give 0.4 g (20% yield) of the title compound as anoff-white solid.

5-Methylaminosulfonyl-2-oxindole

A suspension of 3.38 g of 5-chlorosulfonyl-2-oxindole in 10 mL 2Mmethylamine in tetrahydrofuran was stirred at room temperature for 4hours during which time a white solid formed. The precipitate wascollected by vacuum filtration, washed twice with 5 mL of water anddried under vacuum at 40° C. overnight to give 3.0 g (88% yield) of5-methylaminosulfonyl-2-oxindole.

5-(4-Trifluoromethylphenylaminosulfonyl)-2-oxindole

A suspension of 2.1 g of 5-chlorosulfonyl-2-oxindole, 1.6 g of4-trifluoromethylaniline and 1.4 g of pyridine in 20 mL ofdichloromethane was stirred at room temperature for 4 hours. Theprecipitate which formed was collected by vacuum filtration, washedtwice with 5 mL of water and dried under vacuum at 40° C. overnight togive 2.4 g of crude product containing some impurities by thin layerchromatography. The crude product was chromatographed on silica geleluting with ethyl acetate:hexane (1:2) to give 1.2 g (37% yield) of5-(4-trifluoromethylphenyl-aminosulfonyl)-2-oxindole.

5-(Morpholinosulfonyl)-2-oxindole

A suspension of 2.3 g of 5-chlorosulfonyl-2-oxindole and 2.2 g ofmorpholine in 50 mL of dichloromethane was stirred at room temperaturefor 3 hours. The white precipitate was collected by vacuum filtration,washed with ethyl acetate and hexane and dried under vacuum at 40° C.overnight to give 2.1 g (74% yield) of5-(morpholinosulfonyl)-2-oxindole.

6-Trifluoromethyl-2-oxindole

Dimethylsulfoxide (330 mL) was added to 7.9 g of sodium hydride followedby dropwise addition of 43.6 g diethyloxalate. The mixture was heated to100° C. for 1.0 hour and cooled to room temperature.2-Nitro-4-trifluoromethyltoluene (31.3 g) was added, the reactionstirred for 30 minutes at room temperature and then heated to 100° C.for 1 hour. The reaction was cooled and poured into a mixture ofsaturated aqueous ammonium chloride, ethyl acetate and hexane. Theorganic layer was washed with saturated ammonium chloride, water andbrine, dried, and concentrated to give dimethyl2-(2-nitro-4-trifluoromethylphenyl)malonate.

The diester was dissolved in a mixture of 6.4 g of lithium chloride and2.7 mL of water in 100 mL of dimethylsulfoxide and heated to 100° C. for3 hours. The reaction was cooled and poured into a mixture of ethylacetate and brine. The organic phase was washed with brine, dried withsodium sulfate, concentrated and chromatographed on silica gel (10%ethyl acetate in hexane). The fractions containing product wereevaporated to give 25.7 g of methyl2-nitro-4-trifluoromethylphenylacetate.

Methyl 2-nitro-4-trifluoromethylphenylacetate (26 mg) was hydrogenatedover 10% palladium on carbon and then heated at 100° C. for 3 hours. Thecatalyst was removed by filtration and the solvent evaporated to givethe title compound.

5-(2-Chloroethyl)oxindole

5-Chloroacetyl-2-oxindole(4.18 g) in 30 mL of trifluoroacetic acid in anice bath was treated with 4.65 g of triethylsilane and stirred at roomtemperature for 3 hours. The mixture was poured into 150 mL of water andthe precipitate collected by vacuum filtration, washed with 50 mL ofwater and dried to give 2.53 g (65% yield) of5-(2-chloroethyl)-2-oxindole as a reddish-brown solid.

5-Methoxycarbonyl-2-oxindole

5-Iodo-2-oxindole (17 g) was refluxed with 2 g of palladium diacetate,18.2 g of triethylamine, 150 mL of methanol, 15 mL of dimethylsulfoxideand 2.6 g of DPPP in an atmosphere saturated with carbon monoxide. After24 hours, the reaction was filtered to remove the catalyst and thefiltrate concentrated. The concentrate was chromatographed on silica gel(30% ethyl acetate in hexane). The fractions containing product wereconcentrated and allowed to stand. The precipitated product wascollected by vacuum filtration to give 0.8 g (7%) of the title compoundas an off-white solid.

4-Carboxy-2-oxindole

A solution of trimethylsilyldiazomethane in hexane (2M) was addeddropwise to a solution of 2.01 g of 2-chloro-3-carboxynitrobenzene in 20mL methanol at room temperature until no further gas evolution occurred.The excess trimethylsilyldiazo-methane was quenched with acetic acid.The reaction mixture was dried by rotary pump and the residue wasfurther dried in a vacuum oven overnight. The product(2-chloro-3-methoxycarbonylnitrobenzene) was pure enough for thefollowing reaction.

Dimethyl malonate (6.0 mL) was added to an ice-cold suspension of 2.1 gof sodium hydride in 15 mL of DMSO. The reaction mixture was thenstirred at 100° C. for 1.0 h and then cooled to room temperature.2-Chloro-3-methoxycarbonyl-nitrobenzene (2.15 g) was added to the abovemixture in one portion and the mixture was heated to 100° C. for 1.5 h.The reaction mixture was then cooled to room temperature and poured intoice water, acidified to pH 5, and extracted with ethyl acetate. Theorganic layer was washed with brine, dried over anhydrous sodium sulfateand concentrated to give 3.0 g of the dimethyl2-methoxycarbonyl-6-nitrophenylmalonate.

Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0 g) was refluxed in50 mL of 6 N hydrochloric acid overnight. The mixture was concentratedto dryness and refluxed for 2 hours with 1.1 g of tin(II) chloride in 20mL of ethanol. The mixture was filtered through Celite, concentrated andchromatographed on silica gel (ethyl acetate:hexane:acetic acid) to give0.65 g (37% yield) of 4-carboxy-2-oxindole as a white solid.

5-Carboxy-2-oxindole

2-Oxindole (6.7 g) was added to a stirred suspension of 23 g of aluminumchloride in 30 mL of dichloroethane in an ice bath. Chloroacetylchloride (11.3 g) was slowly added and hydrogen chloride gas wasevolved. After ten minutes of stirring, the reaction was warmed at 40 to50° C. for 1.5 hours. Thin layer chromatography (ethyl acetate, silicagel) showed no remaining starting material. The mixture was cooled toroom temperature and poured into ice water. The precipitate wascollected by vacuum filtration, washed with water and dried under vacuumto give 10.3 g (98%) of 5-chloroacetyl-2-oxindole as an off-white solid.

A suspension of 9.3 g of 5-chloroacetyl-2-oxindole was stirred in 90 mLpyridine at 80 to 90° C. for 3 hours then cooled to room temperature.The precipitate was collected by vacuum filtration and washed with 20 mLethanol. The solid was dissolved in 90 mL 2.5N sodium hydroxide andstirred at 70 to 80° C. for 3 hours. The mixture was cooled to roomtemperature and acidified to pH 2 with 0.5 N hydrochloric acid. Theprecipitate was collected by vacuum filtration and washed thoroughlywith water to give crude 5-carboxy-2-oxindole as a dark brown solid.After standing overnight the filtrate yielded 2 g of5-carboxy-2-oxindole as a yellow solid. The crude dark brown product wasdissolved in hot methanol, the insoluble material removed by filtrationand the filtrate concentrated to give 5.6 g of 5-carboxy-2-oxindole as abrown solid. The combined yield was 97%.

5-Carboxyethyl-2-oxindole

5-Cyanoethyl-2-oxindole (4.02 g) in 10 mL of water containing 25 mL ofconcentrated hydrochloric acid was refluxed for 4 hours. The mixture wascooled, water added and the resulting solid collected by vacuumfiltration, washed with water and dried to give 1.9 g (44% yield) of thetitle compound as a yellow solid.

5-Iodo-4-methyl-2-oxindole

To 2 g of 4-methyl-2-oxindole in 40 mL of glacial acetic acid in an icebath was added 3.67 g N-iodosuccinimide. The mixture was stirred for 1hour, diluted with 100 mL 50% acetic acid in water and filtered. Theresulting white solid was dried under high vacuum to give 3.27 g (88%yield) of the title compound as an off-white solid.

5-Chloro-4-methyl-2-oxindole

A suspension of 3.0 g of 4-methyl-2-oxindole was stirred in 50 mL ofacetonitrile at room temperature while 3.3 g of N-chlorosuccinimide wasadded in portions. Trifluoroacetic acid (1 mL) was then added. Thesuspension was stirred at room temperature for 3 days during which timesolid was always present. The white solid was collected by vacuumfiltration, washed with a small amount of cold acetone and driedovernight in a vacuum oven at 40° C. to give 2.5 g (68%) of5-chloro-4-methyl-2-oxindole.

5-Butyl-2-oxindole

Triethylsilane (2.3 g) was added to 2 g 4-butanoyl-2-oxindole in 20 mLof trifluoroacetic acid at room temperature and the solution stirred for3 hours. The reaction was poured into ice water to give a red oil whichsolidified after standing. The solid was collected by vacuum filtration,washed with water and hexane and dried to give 1.7 g (91% yield) of thetitle compound as an off-white solid.

5-Ethyl-2-oxindole

To 5-Acetyl-2-oxindole (2 g) in 15 mL of trifluoroacetic acid in an icebath was slowly added 1.8 g of triethylsilane; the reaction was thenstirred at room temperature for 5 hours. One mL of triethylsilane wasadded and the stirring continued overnight. The reaction mixture waspoured into ice water and the resulting precipitate collected by vacuumfiltration, washed copiously with water and dried under vacuum to give1.3 g (71% yield) of the title compound as a yellow solid.

5-(Morpholin-4-ethyl)-2-oxindole

5-Chloroethyl-2-oxindole (2.3 g), 1.2 mL of morpholine and 1.2 mL ofdiisopropylethylamine were heated overnight at 100° C. in 10 mL ofdimethylsulfoxide. The mixture ws cooled, poured into water and extactedwith ethyl acetate. The organic layer was washed with brine, dried andevaporated. The residue was chromatographed on silica gel (5% methanolin chloroform) to give 0.9 g (31%) of the title compound as a whitesolid.

5-(4-Methoxycarbonylbenzamido)-2-oxindole

A mixture of 82.0 mg 5-amino-2-oxindole and 131.0 mg4-methoxycarbonylbenzoyl chloride in pyridine was stirred at roomtemperature for 3 hr and poured into ice water. The precipitate wasfiltered, washed with water and dried in a vacuum oven to give 138.0 mgof 5-(4-methoxycarbonylbenzamido)-2-oxindole (81% yield).

5-(4-Carboxybenzamido)-2-oxindole

5-(4-Methoxycarbonylbenzamido)-2-oxindole (0.9 g) and 0.4 g of sodiumhydroxide in 25 mL of methanol were refluxed for 3 hours. The mixturewas concentrated, water added, and the mixture acidified with 6Nhydrochloric acid. The precipitate was collected by vacuum filtration togive 0.75 g (87%) of the title compound as a white solid.

5-Methoxy-2-oxindole

Chloral hydrate (9.6 g) was dissolved in 200 mL of water containing 83 gof sodium sulfate. The solution was warmed to 60° C., a solution of 11.4g of hydroxylamine hydrochloride in 50 mL of water was added and themixture was held at 60° C. In a separate flask, 6.4 g of 4-anisidine and4.3 mL of concentrated hydrochloric acid in 80 mL of water was warmed to80° C. The first solution was added to the second and the mixturerefluxed for 2 minutes after which it was cooled slowly to roomtemperature and then cooled in an ice bath. The tan precipitate wascollected by vacuum filtration, washed with water and dried under vacuumto give 8.6 g (85% yield) of N-(2-hydroximino-acetyl)anisidine.

Concentrated sulfuric acid (45 mL) containing 5 mL of water was warmedto 60° C. and 8.6 g of N-(2-hydroximinoacetyl)anisidine was added in oneportion. The stirred mixture was heated to 93° C. for 10 minutes andthen allowed to cool to room temperature. The mixture was poured into500 g of ice and extracted 3 times with ethyl acetate. The combinedextracts were dried over anhydrous sodium sulfate and concentrated togive 5.1 g (65% yield) of 5-methoxyisatin as a dark red solid.5-Methoxyisatin (5.0 g) and 30 mL of hydrazine hydrate were heated toreflux for 15 minutes. The reaction mixture was cooled to roomtemperature and 50 mL of water was added. The mixture was extracted 3times with 25 mL of ethyl acetate each time, the organic layerscombined, dried over anhydrous sodium sulfate and concentrated to give ayellow solid. The solid was stirred in ethyl acetate and 1.1 g ofinsoluble material was removed by vacuum filtration and saved. Thismaterial proved to be 2-hydrazinocarbonylmethyl-4-anisidine. Thefiltrate was concentrated and chromatographed on silica gel eluting withethyl acetate:hexane (1:1) to give 0.7 g of 5-methoxy-2-oxindole as ayellow solid. The 1.1 g of 2-hydrazino-carbonylmethyl-4-anisidine wasrefluxed for 1 hour in 20 mL of 1N sodium hydroxide. The mixture wascooled, acidified to pH 2 with concentrated hydrochloric acid andextracted 3 times with 25 mL of ethyl acetate each time. The organicextracts were combined, washed with brine, dried over anhydrous sodiumsulfate and concentrated to give 0.8 g of 5-methoxy-2-oxindole as ayellow solid. The combined yield was 1.5 g or 33%.

7-Azaoxindole

3,3-Dibromo-7-azaoxindole (2.9 g) was dissolved in a mixture of 20 mL ofacetic acid and 30 mL of acetonitrile. To the solution was added 6.5 gof zinc dust. The mixture was stirred for 2 hrs at room temperature. Thesolid was filtered from the mixture and the solvent evaporated. Theresidue was slurried with ethyl acetate. The ethyl acetate solutioncontaining insoluble solid was passed through a short column of silicagel. The collected ethyl acetate solution was evaporated and the residuedried under vacuum to give 1.8 g (yield 91%) of 7-azaoxindole aceticacid salt.

5-Dimethylaminosulfonyl-2-oxindole

A suspension of 2.3 g 5-chlorosulfonyl-2-oxindole in 10 mL 2Mdimethylamine in methanol was stirred at room temperature for 4 hours atwhich time a white solid formed. The precipitate was collected by vacuumfiltration, washed with 5 mL of 1N sodium hydroxide and 5 mL of 1Nhydrochloric acid and dried under vacuum at 40° C. overnight to give 1.9g (79% yield) of 5-dimethylamino-sulfonyl-2-oxindole.

6-Phenyl-2-oxindole

Dimethyl malonate (10 mL) in 25 mL of dimethylsulfoxide was addeddropwise to 3.5 g sodium hydride suspended in 25 mL dimethylsulfoxideand the mixture heated at 100° C. for 10 minutes. The mixture was cooledto room temperature and 4.7 g of 4-fluoro-3-nitrobiphenyl in 25 mLdimethylsulfoxide was added. The mixture was heated at 100° C. for 2hours, cooled and quenched with 300 mL of saturated ammonium chloridesolution. The mixture was extracted three times with ethyl acetate andthe combined organic layers washed with water and brine and evaporatedto give, as a yellow oil, crude dimethyl-3-nitrobiphenyl-4-malonate.

Crude dimethyl-3-nitrobiphenyl-4-malonate was refluxed in 30 mL of 6 Nhydrochloric acid for 24 hours. The precipitate was collected byfiltration, washed with water and dried to give 4.5 g of3-nitrobiphenyl-4-acetic acid as a cream colored solid.

Iron powder (2.6 g) was added all at once to 4.5 g of3-nitrobiphenyl-4-acetic acid in 40 mL of acetic acid. The mixture wasrefluxed for 2 hours, concentrated to dryness and taken up in ethylacetate. The solids were removed by filtration and the filtrate washedtwice with 1N hydrochloric acid and brine and dried over anhydroussodium sulfate. The filtrate was concentrated to give 3.4 g (93% yield)of 6-phenyl-2-oxindole as a light brown solid.

6-(2-Methoxyphenyl)-2-oxindole

Tetrakis(triphenylphosphine)palladium (I g) was added to a mixture of 5g 2-methoxyphenylboronic acid, 6.6 g 5-bromo-2-fluoronitrobenzene and 30mL of 2 M sodium carbonate solution in 50 mL of toluene and 50 mL ofethanol. The mixture was refluxed for 2 hours, concentrated, and theresidue extracted twice with ethyl acetate. The ethyl acetate layer waswashed with water and brine, then dried, and concentrated to give a darkgreen oil which solidified on standing, crude4-fluoro-2′-methoxy-3-nitrobiphenyl.

Dimethyl malonate (14 mL) was added dropwise to 2.9 g of sodium hydridesuspended in 50 mL of dimethylsulfoxide. The mixture was heated at 100°C. for 15 minutes and cooled to room temperature. Crude4-fluoro-2′-methoxy-3-nitrobiphenyl in 60 mL of dimethylsulfoxide wasadded and the mixture was heated at 100° C. for 2 hours. The reactionmixture was cooled and quenched with 300 mL of saturated sodium chloridesolution and extracted twice with ethyl acetate. The extracts werecombined, washed with saturated ammonium chloride, water and brine,dried over anhydrous sodium sulfate and concentrated to give crudedimethyl 2′-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil.

Crude dimethyl 2′-methoxy-3-nitrobiphenyl-4-malonate was heated at 100°C. in 50 mL of 6 N hydrochloric acid for 24 hours and cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 9.8 of 2′-methoxy-2-nitrobiphenyl-4 acetic acid as alight tan solid.

Iron powder (5 g) was added in one portion to 9.8 g of2′-methoxy-3-nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acidwas heated to 100° C. for 3 hours. The reaction mixture was concentratedto dryness, sonicated in ethyl acetate and filtered to remove theinsolubles. The filtrate was washed twice with 1N hydrochloric acid,water and then brine, dried over anhydrous sodium sulfate andconcentrated. The residue was chromatographed on silica gel in ethylacetate:hexane (1:2) to give 5.4 g of 6-(2-methoxyphenyl)-2-oxindole asa rose colored solid.

6-(3-Methoxyphenyl)-2-oxindole

Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of5 g 3-methoxyphenylboronic acid, 5 g 5-bromo-2-fluoro-nitrobenzene and11 mL of 2 M sodium carbonate solution in 100 mL of toluene. The mixturewas refluxed for 2 hours, diluted with water and extracted with ethylacetate. The ethyl acetate was washed with saturated sodium bicarbonateand brine and then dried and concentrated to give an oily solid. Thesolid was chromatographed on silica gel (ethyl acetate:hexane (1:6)) togive 4.3 g (77% yield) of 4-fluoro-3′-methoxy-3-nitrobiphenyl.

Dimethyl malonate (9.7 mL) was added dropwise to 2.0 g sodium hydridesuspended in 50 mL dimethylsulfoxide. The mixture was heated to 100° C.for 35 minutes and cooled to room temperature.4-Fluoro-2′-methoxy-3-nitrobiphenyl (4.2 g) in 50 mL dimethylsulfoxidewas added and the mixture was heated at 100° C. for 1 hour. The reactionmixture was cooled and quenched with 300 mL of saturated ammoniumchloride solution and extracted twice with ethyl acetate. The extractswere combined, washed with brine, dried over anhydrous sodium sulfateand concentrated to give crude dimethyl3′-methoxy-3-nitrobiphenyl-4-malonate as a pale yellow solid.

Crude dimethyl 3′-methoxy-3-nitro-biphenyl-4-malonate was heated at 110°C. in 45 mL 6N hydrochloric acid for 4 days and then cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 5.3 g of 3′-methoxy-2-nitrobiphenyl-4-acetic acid as alight tan solid.

3′-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was dissolved inmethanol and hydrogenated over 0.8 g of 10% palladium on carbon for 3hours at room temperature. The catalyst was removed by filtration,washed with methanol and the filtrates combined and concentrated to givea brown solid. The solid was chromatographed on silica gel in ethylacetate:hexane:acetic acid (33:66:1) to give 3.0 g of6-(3-methoxypheny)-2-oxindole as a pink solid.

6-(4-Methoxyphenyl)-2-oxindole

Tetrakis(triphenylphosphine)palladium (I g) was added to a mixture of 5g of 4methoxyphenylboronic acid, 6.6 g of 5-bromo-2-fluoronitrobenzeneand 30 mL of 2 M sodium carbonate solution in 50 mL of toluene and 50 mLof ethanol. The mixture was refluxed for 2 hours, concentrated, and theresidue extracted twice with ethyl acetate. The ethyl acetate layer waswashed with water and brine, dried, and concentrated to give a brownoily solid. The solid was chromatographed on silica gel (5% ethylacetate in hexane) to give crude 4-fluoro-4′-methoxy-3-nitrobiphenyl asa pale yellow solid.

Dimethyl malonate (10 mL) was added dropwise to 2.0 g of sodium hydridesuspended in 60 mL of dimethylsulfoxide. The mixture was heated to 100°C. for 10 minutes and cooled to room temperature. Crude4-fluoro-2′-methoxy-3-nitrobiphenyl (5.2 g) in 50 mL dimethylsulfoxidewas added and the mixture was heated at 100° C. for 2 hours. Thereaction mixture was cooled and quenched with 300 mL of saturated sodiumchloride solution and extracted three times with ethyl acetate. Theextracts were combined, washed with saturated ammonium chloride, waterand brine, dried over anhydrous sodium sulfate and concentrated to givecrude dimethyl 4′-methoxy-3-nitrobiphenyl-4 malonate as a yellow oil.

Crude dimethyl 4′-methoxy-3-nitro-biphenyl-4-malonate was heated at 100°C. in 60 mL of 6N hydrochloric acid for 15 hours and cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 7.2 g of crude 4′-methoxy-3-nitrobiphenyl-4-aceticacid as a light tan solid.

Iron powder (3.6 g) was added in one portion to 7.2 g of4′-methoxy-3-nitrobiphenyl-4-acetic acid in 50 mL of glacial acetic acidand heated at 100° C. overnight. The reaction mixture was concentratedto dryness, sonicated in ethyl acetate and filtered to remove theinsolubles. The filtrate was washed twice with 1N hydrochloric acid andbrine, dried over anhydrous sodium sulfate and concentrated to give 2.7g of 6-(4-methoxyphenyl)-2-oxindole as a rose colored solid.

6-(3-Ethoxyphenyl)-2-oxindole

Tetrakis(triphenylphosphine)palladium (0.8 g) was added to a mixture of4.2 g of 3-ethoxyphenylboronic acid, 5.0 g of5-bromo-2-fluoronitrobenzene and 22 mL of 2 M sodium carbonate solutionin 50 mL of toluene and 50 mL of ethanol. The mixture was refluxed for 2hours, concentrated, water was added and the mixture was extracted twicewith ethyl acetate. The ethyl acetate layer was washed with water andbrine, then dried, and concentrated. The residue was chromatographed onsilica gel (5% ethyl acetate in hexane) to give 5.3 g (90% yield) ofcrude 4-fluoro-3′-ethoxy-3-nitrobiphenyl as a yellow oil.

Dimethyl malonate (11.4 mL) was added dropwise to 4.0 g sodium hydridesuspended in 20 mL dimethylsulfoxide. The mixture was heated to 100° C.for 10 minutes and then cooled to room temperature. Crude4-fluoro-3′-ethoxy-3-nitrobiphenyl (5.3 g) in 25 mL of dimethylsulfoxidewas added and the mixture was heated at 100° C. for 2 hours. Thereaction mixture was cooled and quenched with 300 mL of saturatedammonium chloride solution and extracted three times with ethyl acetate.The extracts were combined, washed with water and brine and then driedover anhydrous sodium sulfate and concentrated to give crude dimethyl3′-ethoxy-3-nitrobiphenyl-4-malonate as a yellow oil.

Crude dimethyl 3′-ethoxy-3-nitrobiphenyl-4-malonate was heated at 100°C. in 60 mL of 6N hydrochloric acid for 4 days and then cooled. Theprecipitate was collected by filtration, washed with water and hexane,and dried to give 4.7 g of crude 3′-ethoxy-3-nitrobiphenyl-4-acetic acidas a light tan solid.

Iron powder (2.4 g) was added in one portion to 4.6 g of3′-ethoxy-3-nitrobiphenyl-4-acetic acid in 40 mL of glacial acetic acidand refluxed for 2 hours. The reaction mixture was concentrated todryness, treated repeatedly with ethyl acetate and filtered to removethe insolubles. The filtrate was washed twice with 1N hydrochloric acidand brine and then dried over anhydrous sodium sulfate and concentratedto give 3.5 g (91% yield) of 6-(3-ethoxyphenyl)-2-oxindole as a lightbrown solid.

6-Bromo-2-oxindole

Dimethyl malonate (13 mL) was added dropwise to 2.7 g sodium hydridesuspended in 20 mL dimethylsulfoxide. The mixture was heated to 100° C.for 10 minutes and then cooled to room temperature.5-Bromo-2-fluoronitrobenzene (5.0 g) in 25 mL of dimethylsulfoxide wasadded and the mixture was heated at 100° C. for 2 hours. The reactionmixture was cooled and quenched with 300 mL of saturated ammoniumchloride solution and extracted three times with ethyl acetate. Theextracts were combined, washed with saturated ammonium chloride, waterand brine, dried over anhydrous sodium sulfate and concentrated to givecrude dimethyl 4-bromo-2-nitrophenylmalonate as a pale yellow oil.

Crude dimethyl 4-bromo-2-nitrophenylmalonate was heated at 110° C. in 40mL of 6N hydrochloric acid for 24 hours and then cooled. The precipitatewas collected by filtration, washed with water and dried to give 5.3 g(89% yield) of 4-bromo-2-nitro-phenylacetic acid as an off white solid.

4-Bromo-2-nitrophenylacetic acid (0.26 g), 0.26 g zinc powder and 3 mL50% sulfuric acid in 5 mL of ethanol were heated at 100° C. overnight.The reaction mixture was filtered, diluted with a little acetic acid,concentrated to remove ethanol, diluted with water and extracted twicewith ethyl acetate. The combined extracts were washed with brine, driedover anhydrous sodium sulfate and concentrated to give 0.19 g (90%yield) of 6-bromo-2-oxindole as a yellow solid.

5-Acetyl-2-oxindole

2-Oxindole (3 g) was suspended in 1,2-dichloroethane and 3.2 mL acetylchloride were slowly added. The resulting suspension was heated to 50°C. for 5 hours, cooled, and poured into water. The resulting precipitatewas collected by vacuum filtration, washed copiously with water anddried under vacuum to give 2.9 g (73% yield) of the title compound as abrown solid.

5-Butanoyl-2-oxindole

To 15 g aluminum chloride suspended in 30 mL 1,2-dichloro-ethane in anice bath was added 7.5 g of 2-oxindole and then 12 g of butanoylchloride. The resulting suspension was heated to 50° C. overnight. Themixture was poured into ice water and extracted 3 times with ethylacetate. The combined ethyl acetate layers were washed with brine, driedover sodium sulfate, and concentrated to dryness to give a brown solid.The solid was chromatographed on silica gel (50% ethyl acetate inhexane) to give 3 g (25%) of the title compound as a yellow solid.

5-Cyanoethyl-2-oxindole

Potassium cyanide (2.0 g) was added to 15 mL of dimethyl-sulfoxide andheated to 90° C. 5-Chloroethyl-2-oxindole (3.0 g) dissolved in 5 mLdimethyl sulfoxide was added slowly with stirring, and the reactionheated to 150° C. for 2 hours. The mixture was cooled, poured into icewater and the precipitate collected by vacuum filtration, washed withwater, dried and then chromatographed on silica gel (5% methanol inchloroform) to give 1.2 g (42% yield) of the title compound.

6-Morpholin-4-yl)-2-oxindole

6-Amino-2-oxindole (2.2 g), 4.0 g 2, 2′-dibromoethyl ether and 7.9 gsodium carbonate were refluxed in 20 ml ethanol overnight, concentratedand diluted with 50 ml of water. The mixture was extracted three timeswith 50 ml of ethyl acetate and the organic extracts combined, washedwith 20 ml of brine, dried over anhydrous sodium sulfate andconcentrated to dryness. The solid was chromatographed on a column ofsilica gel (ethyl acetate:hexane (1:1) containing 0.7% acetic acid) togive 1.2 g (37% yield) of the title compound as a beige solid.

6-(3-Trifluoroacetylphenyl)-2-oxindole

3-Aminophenylboronic acid (3.9 g), 5 g 5-bromo-2-fluoro-nitrobenzene,0.8 g tetrakis(triphenylphosphine)palladium and 23 mL of 2 M sodiumbicarbonate solution in 50 mL of toluene were refluxed under nitrogenfor 2.5 hours. The reaction mixture was poured into 200 mL of ice waterand the mixture extracted three times with 50 mL of ethyl acetate. Thecombined organic layers were washed with 50 mL of water and 20 mL ofbrine, dried over, anhydrous sodium sulfate and concentrated to give 9.7g (92% yield) of 2-fluoro-5-(3-aminophenyl)nitrobenzene as a dark brownoil.

Trifluoroacetic anhydride (5.4 mL) was slowly added to a stirredsolution of 9.7 g 2-fluoro-5-(3-aminophenyl)-nitrobenzene and 5.3 mL oftriethylamine in 50 mL of dichloromethane at 0° C. and the mixture wasstirred for an additional 20 minutes. The mixture was concentrated andthe residue chromatographed on a column of silica gel (10% ethyl acetatein hexane) to give 8.6 g (65% yield) of2-fluoro-5-(3-trifluoroacetamidophenyl)nitrobenzene as a pale orange oilwhich solidified on standing.

Dimethyl malonate (9.6 mL) was added dropwise to a stirred suspension of3.2 g of 60% sodium hydride in mineral oil in 40 mL anhydrousdimethylsulfoxide under nitrogen. The mixture was stirred for 10 minutesand 2-fluoro-5-(3-trifluoroacetamidophenyl)nitrobenzene in 20 mLdimethylsulfoxide was added. The resulting dark red mixture was heatedto 100° C. for 2 hours. The reaction was quenched by pouring into 100 mLof saturated ammnonium chloride solution and extracted twice with 50 mLof ethyl acetate. The organic phase was washed with 50 mL each ofsaturated ammonium chloride solution, water, and brine, dried overanhydrous sodium sulfate and concentrated to a yellow oil. The oil waschromatographed on a column of silica gel (ethyl acetate:hexane (1:4))to give 4.4 g (50% yield) of dimethyl2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl] malonate as a paleyellow solid.

Dimethyl 2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl]-malonate (4.4g) was refluxed overnight in 50 mL 6N hydrochloric acid. The reactionmixture was cooled to room temperature and the solids were collected byvacuum filtration, washed with water, and dried under vacuum to give 2.7g (73% yield) of 2-[2-nitro-4-(3-trifluoroacetamidophenyl)phenyl] aceticacid.

2-[2-Nitro-4-(3-trifluoroacetamidophenyl)phenyl]acetic acid (100 mg) and50 mg iron powder in 3 mL acetic acid was heated at 100° C. for 2 hours.The reaction mixture was concentrated and the residue sonicated in 5 mLethyl acetate. The insoluble solids were removed by vacuum filtrationand the filtrate washed with 1N hydrochloric acid, water and brine,dried over anhydrous sodium sulfate and concentrated to give 10 mg (14%yield) of the title compound as a rose-colored solid.

5. Biological Evaluation

It will be appreciated that, in any given series of compounds, aspectrum of biological activity will be afforded. In its most preferredembodiments, this invention relates to novel3-heteroarylidenyl-2-indolinones having improved hydrosolubility anddemonstrating the ability to modulate RTK and CTK activity. Thefollowing assays are employed to select those compounds demonstratingthe optimal degree of the desired activity.

As used herein, the phrase “optimal degree of the desired activity”refers to the lowest IC50, defined elsewhere herein, against a PTKrelated to a particular disorder so as to provide an organism,preferably a human, with a therapeutically effective amount of acompound of this invention at the lowest possible dosage.

B. Assay Procedures.

The following in vitro assays may be used to determine the level ofactivity and effect of the different compounds of the present inventionon one or more of the RTKs. Similar assays can be designed along thesame lines for any PTK using techniques well known in the art.

The cellular/catalytic assays described herein are performed in an ELISAformat. The general procedure is as follows: a compound is introduced tocells expressing the test kinase, either naturally or recombinantly, forsome period of time after which, if the test kinase is a receptor, aligand known to activate the receptor's activity is added. The cells arelysed and the lysate is transferred to the wells of an ELISA platepreviously coated with a specific antibody recognizing the substrate ofthe enzymatic phosphorylation reaction. Non-substrate components of thecell lysate are washed away and the amount of phosphorylation on thesubstrate is detected with an antibody specifically recognizingphosphotyrosine compared with control cells that were not contacted witha test compound.

The cellular/biologic assays described herein measure the amount of DNAmade in response to activation of a test kinase, which is a generalmeasure of a proliferative response. The general procedure for thisassay is as follows: a compound is introduced to cells expressing thetest kinase, either naturally or recombinantly, for some period of timeafter which, if the test kinase is a receptor, a ligand known toactivate the receptor's activity is added. After incubation at leastovernight, a DNA labeling reagent such as Bromodeoxy-uridine (BrdU) or3H-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

1. Cellular/Catalytic Assays

Enzyme linked immunosorbent assays (ELISA) may be used to detect andmeasure the presence of PTK activity. The ELISA may be conductedaccording to known protocols which are described in, for example,Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In: Manual ofClinical Immunology, 2d ed., edited by Rose and Friedman, pp 359-371 Am.Soc. Of Microbiology, Washington, D.C.

The disclosed protocol may be adapted for determining activity withrespect to a specific RTK. For example, the preferred protocols forconducting the ELISA experiments for specific RTKs is provided below.Adaptation of these protocols for determining a compound's activity forother members of the RTK family, as well as for CTKs, is well within thescope of knowledge of those skilled in the art.

a. FLK-1

An ELISA assay is conducted to measure the kinase activity of the FLK-1receptor and more specifically, the inhibition or activation of TKactivity on the FLK-1 receptor. Specifically, the following assay can beconducted to measure kinase activity of the FLK-1 receptor in cellsgenetically engineered to express Flk-1.

Materials And Methods.

Materials. The following reagents and supplies are used:

a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);

b. Cappel goat anti-rabbit IgG (catalog no. 55641);

c. PBS (Gibco Catalog No. 450-1300EB);

d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);

e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);

f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% TritonX-100, and 10% glycerol);

g. EDTA (0.5 M (pH 7.0) as a 100×stock);

h. Sodium orthovanadate (0.5 M as a 100×stock);

i. Sodium pyrophosphate (0.2 M as a 100×stock);

j. NUNC 96 well V bottom polypropylene plates (Applied ScientificCatalog No. AS-72092);

k. NIH3T3 C7#3 Cells (FLK-1 expressing cells);

l. DMEM with 1X high glucose L-Glutamine (catalog No. 11965-050);

m. FBS, Gibco (catalog no. 16000-028);

n. L-glutamine, Gibco (catalog no. 25030-016);

o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 μg/100 μl stockin Milli-Q dH₂O and stored at −20° C.;

p. Affinity purified anti-FLK-l antiserum;

q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley,et al., 1990, Cancer Research 50:1550-1558);

r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);

s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution(100 mM citric acid (anhydrous), 250 mM Na₂HPO₄ (pH 4.0), 0.5 mg/ml ABTS(Sigma catalog no. A-1888)), solution should be stored in dark at 4° C.until ready for use;

t. H₂O₂ (30% solution) (Fisher catalog no. H325)

u. ABTS/H₂O (15 ml ABTS solution, 2 μl H₂O₂) prepared 5 minutes beforeuse and left at room temperature;

v. 0.2 M HCl stock in H₂O;

w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418); and

y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).

Protocol. The following protocol can be used for conducting the assay:

1. Coat Corning 96-well ELISA plates with 1.0 μg per well CappelAnti-rabbit IgG antibody in 0.1M Na₂CO₃ pH 9.6. Bring final volume to150 μl per well. Coat plates overnight at 4° C. Plates can be kept up totwo weeks when stored at 4° C.

2. Grow cells in Growth media(DMEM, supplemented with 2.0 mML-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37°C., 5% CO₂.

3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene96-well round bottom cell plates, 25.000 cells/well in 200 μl of growthmedia.

4. Grow cells at least one day at 37° C., 5% CO₂.

5. Wash cells with D-PBS 1X.

6. Add 200 μl/well of starvation media (DMEM, 2.0 mM 1-Glutamine, 0.1%FBS). Incubate overnight at 37° C., 5% CO₂.

7. Dilute Compounds 1:20 in polypropylene 96 well plates usingstarvation media. Dilute dimethylsulfoxide 1:20 for use in controlwells.

8. Remove starvation media from 96 well cell culture plates and add 162μl of fresh starvation media to each well.

9. Add 18 μl of 1:20 diluted Compound dilution (from step 7) to eachwell plus the 1:20 dimethylsulfoxide dilution to the control wells (+/−VEGF), for a final dilution of 1:200 after cell stimulation. Finaldimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO₂ for twohours.

10. Remove unbound antibody from ELISA plates by inverting plate toremove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Pat theplate on a paper towel to remove excess liquid and bubbles.

11. Block plates with TBSW+0.5% Ethanolamine, pH 7.0, 150 μl per well.Incubate plate thirty minutes while shaking on a microtiter plateshaker.

12. Wash plate 3 times as described in step 10.

13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbitantiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolaminepH 7.0. Incubate plate for thirty minutes while shaking.

14. Add 180 μl starvation medium to the cells and stimulate cells with20 μl/well 10.0 mM sodium ortho vanadate and 500 ng/ml VEGF (resultingin a final concentration of 1.0 mM sodium ortho vanadate and 50 ng/mlVEGF per well) for eight minutes at 37° C., 5% CO₂. Negative controlwells receive only starvation medium.

15. After eight minutes, media should be removed from the cells andwashed one time with 200 μl/well PBS.

16. Lyse cells in 150 μl/well HNTG while shaking at room temperature forfive minutes. HNTG formulation includes sodium ortho vanadate, sodiumpyrophosphate and EDTA.

17. Wash ELISA plate three times as described in step 10.

18. Transfer cell lysates from the cell plate to ELISA plate andincubate while shaking for two hours. To transfer cell lysate pipette upand down while scrapping the wells.

19. Wash plate three times as described in step 10.

20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05%ethanolamine. Bring final volume to 150 μl/well. Incubate while shakingfor 30 minutes.

21. Wash plate three times as described in step 10.

22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouseIgG conjugated horseradish peroxidase in TBSW+0.5% ethanolamine, pH 7.0.Bring final volume to 150 μl/well. Incubate while shaking for thirtyminutes.

23. Wash plate as described in step 10.

24. Add 100 μl of ABTS/H₂O₂ solution to well. Incubate ten minutes whileshaking.

25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final to stop the colordevelopment reaction. Shake 1 minute at room temperature. Remove bubbleswith slow stream of air and read the ELISA plate in an ELISA platereader at 410 nm.

b. HER-2 ELISA

Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells

HER2 kinase activity in whole EGFR-NIH3T3 cells are measured asdescribed below:

Materials and Reagents. The following materials and reagents can be usedto conduct the assay:

a. EGF: stock concentration: 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley,et al., supra).

d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer:

Tris-HCl, pH 7.2  50 mM NaCl 150 mM Triton X-100 0.1

f. HNTG 5X stock:

HEPES  0.1 M NaCl 0.75 M Glycerol 50% Triton X-100 1.0%

g. ABTS stock:

Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc.  0.5 pM ABTS  0.5 mg/ml*(2,2′-azinobis(3-ethylbenzthiazolinesuifonic acid)). Keep solution indark at 4° C. until use.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄(P₂O₇) 0.2 M

Procedure. The following protocol is used:

A. Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101antibody at 0.5 g per well in PBS, 100 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with 100 μl blockingbuffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate theplate, shaking, at room temperature (about 23° C. to 25° C.) for 30minutes. Just prior to use, remove blocking buffer and wash plate 4times with TBST buffer.

B. Seeding Cells

1. An NIH3T3 cell line overexpressing a chimeric receptor containing theEGFR extracellular domain and intracellular HER2 kinase domain can beused for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% fetal bovine serum. Suspend cellsin DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, atroom temperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

C. Assay Procedures

1. Check seeded cells for contamination using an inverted microscope.Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer5 μl to a TBST well for a final drug dilution of 1:200 and a final DMSOconcentration of 1%. Control wells receive DMSO alone. Incubate in 5%CO₂ at 37° C. for two hours.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 100 nM final concentration isattained.

3. Prepare fresh HNTG* sufficient for 100 μl per well; and place on ice.

HNTG* (10 ml): HNTG stock 2.0 ml milli-Q H₂O 7.3 ml EDTA, 100 mM, pH 7.00.5 ml Na₃VO₄, 0.5 M 0.1 ml Na₄(P₂O₇), 0.2 M 0.1 ml

4. After 120 minutes incubation with drug, add prepared SGF ligand tocells, 10 μl per well, to a final concentration of 100 nM. Control wellsreceive DMEM alone. Incubate, shaking, at room temperature, for 5minutes.

5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTGto cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, removeblocking buffer from other ELISA plate and wash with TBST as describedabove.

6. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG^(*) lysis buffer. Transfer lysate to a coated,blocked, and washed ELISA plate. Incubate shaking at room temperaturefor one hour.

7. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000 dilution in TBST).

9. Remove TAGO detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate shaking at room temperature for 20 minutes. (ABTS/H₂O₂solution: 1.0 μl 30% H₂O₂ in 10 ml ABTS stock).

10. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

11. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

C. PDGF-R ELISA

All cell culture media, glutamine, and fetal bovine serum can bepurchased from Gibco Life Technologies (Grand Island, N.Y.) unlessotherwise specified. All cells are grown in a humid atmosphere of 90-95%air and 5-10% CO₂ at 37° C. All cell lines are routinely subculturedtwice a week and are negative for mycoplasma as determined by theMycotect method (Gibco).

For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU)are grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA,1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in0.5% serum at 25,000 to 30,000 cells per well. After overnightincubation in 0.5% serum-containing medium, cells are changed toserum-free medium and treated with test compound for 2 hr in a 5% CO₂,37° C. incubator. Cells are then stimulated with ligand for 5-10 minutefollowed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5mM EDTA, 5 mM Na₃VO₄, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates(0.5 mg/well in PBS) are transferred to ELISA plates previously coatedwith receptor-specific antibody and which had been blocked with 5% milkin TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) atroom temperature for 30 min. Lysates are incubated with shaking for 1hour at room temperature. The plates are washed with TBST four times andthen incubated with polyclonal anti-phosphotyrosine antibody at roomtemperature for 30 minutes. Excess anti-phosphotyrosine antibody isremoved by rinsing the plate with TBST four times. Goat anti-rabbit IgGantibody is added to the ELISA plate for 30 min at room temperaturefollowed by rinsing with TBST four more times. ABTS (100 mM citric acid,250 mM Na₂HPO₄ and 0.5 mg/mL2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H₂O₂ (1.2 mL30% H₂O₂ to 10 ml ABTS) is added to the ELISA plates to start colordevelopment. Absorbance at 410 nm with a reference wavelength of 630 nmis recorded about 15 to 30 min after ABTS addition.

d. IGF-I RECEPTOR ELISA

The following protocol may be used to measure phosphotyrosine level onIGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity.

Materials And Reagents. The following materials and reagents are used:

a. The cell line used in this assay is 3T3/IGF-1R, a cell linegenetically engineered to overexpresses IGF-1 receptor.

b. NIH3T3/IGF-1R is grown in an incubator with 5% CO₂ at 37° C. Thegrowth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.

c. Affinity purified anti-IGF-1R antibody 17-69.

d. D-PBS:

KH₂PO₄ 0.20 g/l K₂HPO₄ 2.16 g/l KCl 0.20 g/l NaCl 8.00 g/l (pH 7.2)

e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat DryMilk).

f. TBST buffer:

Tris-HCl  50 mM NaCl 150 mM (pH 7.2/HCl 10 N) Triton X-100 0.1%

 Stock solution of TBS (10×) is prepared, and Triton X-100 is added tothe buffer during dilution.

g. HNTG buffer:

HEPES  20 mM NaCl 150 mM (pH 7.2/HCl 1 N) Glycerol 10% Triton X-100 0.2%

 Stock solution (5×) is prepared and kept at 4° C.

h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100×stock.

I. Na₃VO₄:0.5 M as 100×stock and aliquots are kept in −80° C.

j. Na₄P₂O₇:0.2 M as 100×stock.

k. Insulin-like growth factor-1 from Promega (Cat# G5111).

l. Rabbit polyclonal anti-phosphotyrosine antiserum.

m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat.No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif.

n. ABTS (2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution:

Citric acid 100 mM Na₂HPO₄ 250 mM (pH 4.0/1 N HCl) ABTS 0.5 mg/ml

 ABTS solution should be kept in dark and 4° C. The solution should bediscarded when it turns green.

o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C.

Procedure. All the following steps are conducted at room temperatureunless it is specifically indicated. All ELISA plate washings areperformed by rinsing the plate with tap water three times, followed byone TBST rinse. Pat plate dry with paper towels.

A. Cell Seeding:

1. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90%confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100,GIBCO).

2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine, andtransfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000cells/well (100 μl/well). Incubate for 1 day then replace medium toserum-free medium (90/ μl) and incubate in 5% CO₂ and 37° C. overnight.

B. ELISA Plate Coating and Blocking:

1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at0.5 μg/well in 100 μl PBS at least 2 hours.

2. Remove the coating solution, and replace with 100 μl Blocking Buffer,and shake for 30 minutes. Remove the blocking buffer and wash the platejust before adding lysate.

C. Assay Procedures:

1. The drugs are tested in serum-free condition.

2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-wellpoly-propylene plate, and transfer 10 μl/well of this solution to thecells to achieve final drug dilution 1:100, and final DMSO concentrationof 1.0%. Incubate the cells in 5% CO₂ at 37° C. for 2 hours.

3. Prepare fresh cell lysis buffer (HNTG*)

HNTG   2 ml EDTA 0.1 ml Na₃VO₄ 0.1 ml Na₄(P₂O₇) 0.1 ml H₂O 7.3 ml

4. After drug incubation for two hours, transfer 10 μl/well of 200 nMIGF-1 Ligand in PBS to the cells (Final Conc. =20 nM), and incubate at5% CO₂ at 37° C. for 10 minutes.

5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Lookat cells under microscope to see if they are adequately lysed.

6. Use a 12-channel pipette to scrape the cells from the plate, andhomogenize the lysate by repeated aspiration and dispensing. Transferall the lysate to the antibody coated ELISA plate, and shake for 1 hour.

7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 withTEST) 100 μl/well, and shake for 30 minutes.

8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 with TBST)100 μl/well, and shake for 30 minutes.

9. Remove detection antibody, wash the plate, and transfer freshABTS/H₂O₂ (1.2 μl H₂O₂ to 10 ml ABTS) 100 μl/well to the plate to startcolor development.

10. Measure OD at 410 nm with a reference wavelength of 630 nm inDynatec MR5000.

e. EGF Receptor ELISA

EGF Receptor kinase activity in cells genetically engineered to expresshuman EGF-R can be measured as described below:

Materials and Reagents. The following materials and reagents are used

a. EGF Ligand: stock concentration =16.5 μM; EGF 201, TOYOBO, Co., Ltd.Japan.

b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellulardomain).

c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).

d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidaseconjugate, TAGO, Inc., Burlingame, Calif.

e. TBST buffer:

Tris-HCl, pH 7  50 mM NaCl 150 mM Triton X-100 0.1

f. HNTG 5X buffer:

HEPES  0.1 M NaCl 0.75 M Glycerol 50 Triton X-100 1.0%

g. ABTS stock:

Citric Acid 100 mM Na₂HPO₄ 250 mM HCl, conc.  4.0 pH ABTS*  0.5 mg/ml

Keep solution in dark at 4° C. until used.

h. Stock reagents of:

EDTA 100 mM pH 7.0

Na₃VO₄ 0.5 M

Na₄(P₂O₇) 0.2 M

Procedure. The following protocol is used:

A. Pre-coat ELISA Plate

1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and storeovernight at 4° C. Coated plates are good for up to 10 days when storedat 4° C.

2. On day of use, remove coating buffer and replace with blocking buffer(5% Carnation Instant NonFat Dry Milk in PBS). Incubate the plate,shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes.Just prior to use, remove blocking buffer and wash plate 4 times withTBST buffer.

B. Seeding Cells

1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can beuse for this assay.

2. Choose dishes having 80-90% confluence for the experiment. Trypsinizecells and stop reaction by adding 10% CS DMEM medium. Suspend cells inDMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm at roomtemperature for 5 minutes.

3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), andcount the cells using trypan blue. Viability above 90% is acceptable.Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000cells per well, 100 μl per well, in a 96 well microtiter plate. Incubateseeded cells in 5% CO₂ at 37° C. for about 40 hours.

C. Assay Procedures.

1. Check seeded cells for contamination using an inverted microscope.Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer5 μl to a test well for a final drug dilution of 1:200 and a final DMSOconcentration of 1%. Control wells receive DMSO alone. Incubate in 5%CO₂ at 37° C. for one hour.

2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of10 μl dilute EGF (1:12 dilution), 25 nM final concentration is attained.

3. Prepare fresh 10 ml HNTG^(*) sufficient for 100 μl per well whereinHNTG* comprises: HNTG stock (2.0 ml), milli-Q H₂O (7.3 ml), EDTA, 100mM, pH 7.0 (0.5 ml), Na₃VO₄0.5 M (0.1 ml) and Na₄(P₂O₇), 0.2 M (0.1 ml).

4. Place on ice.

5. After two hours incubation with drug, add prepared EGF ligand tocells, 10 μl per well, to yield a final concentration of 25 nM. Controlwells receive DMEM alone. Incubate, shaking, at room temperature, for 5minutes.

6. Remove drug, EGF, and DMEM. Wash cells twice with PBS. TransferHNTG^(*) to cells, 100 μl per well. Place on ice for 5 minutes.Meanwhile, remove blocking buffer from other ELISA plate and wash withTBST as described above.

7. With a pipette tip securely fitted to a micropipettor, scrape cellsfrom plate and homogenize cell material by repeatedly aspirating anddispensing the HNTG^(*) lysis buffer. Transfer lysate to a coated,blocked, and washed ELISA plate. Incubate shaking at room temperaturefor one hour.

8. Remove lysate and wash 4 times with TBST. Transfer freshly dilutedanti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shakingat room temperature for 30 minutes in the presence of the anti-Ptyrantiserum (1:3000 dilution in TBST).

9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transferthe freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plateat 100 μl per well. Incubate shaking at room temperature for 30 minutes(anti-rabbit IgG antibody: 1:3000 dilution in TBST).

10. Remove detection antibody and wash 4 times with TBST. Transferfreshly prepared ABTS/H₂O₂ solution to ELISA plate, 100 μl per well.Incubate at room temperature for 20 minutes. ABTS/H₂O₂ solution: 1.2 μl30% H₂O₂ in 10 ml ABTS stock.

11. Stop reaction by adding 50 μl 5N H₂SO₄ (optional), and determineO.D. at 410 nm.

12. The maximal phosphotyrosine signal is determined by subtracting thevalue of the negative controls from the positive controls. The percentinhibition of phosphotyrosine content for extract-containing wells isthen calculated, after subtraction of the negative controls.

f. Met Autophosphorylation Assay—ELISA

This assay determines Met tyrosine kinase activity by analyzing Metprotein tyrosine kinase levels on the Met receptor.

1. Reagents

a. HNTG (5×stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCl inabout 350 ml dH2O. Adjust pH to 7.2 with HCl or NaOH, add 500 mlglycerol and 10 ml Triton X-100, mix, add dH2O to 1 L total volume. Tomake 1 L of 1×working solution add 200 ml 5×stock solution to 800 mldH2O, check and adjust pH as necessary, store at 4° C.

b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. # 450-1300 EB(1×solution).

c. Blocking Buffer: in 500 ml dH2O place 100 g BSA, 12.1 g Tris-pH7.5,58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.

d. Kinase Buffer: To 500 ml dH2O add 12.1 g TRIS pH7.2, 58.4 g NaCl,40.7 g MgCl₂ and 1.9 g EGTA; bring to 1 L total volume with dH2O.

e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. # P-7626, to 435.5mg, add 100% ethanol to 25 ml total volume, vortex.

f. ATP (Bacterial Source), Sigma Cat. # A-7699, store powder at −20° C.;to make up solution for use, dissolve 3.31 mg in 1 ml dH₂O.

g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, TransductionLaboratories Cat. # E120H.

h. Pierce 1-Step (TM) Turbo TMB-ELISA (3,3′,5,5′-tetramethylbenzidine,Pierce Cat. # 34022.

i. H₂SO₄, add 1 ml conc. (18N) to 35 ml dH2O.

j. TRIS HCL, Fischer Cat. # BP152-5; to 121.14 g of material, add 600 mlMilliQ H₂O, adjust pH to 7.5 (or 7.2) with HCl, bring volume to 1 L withMilliQ H₂O.

k. NaCl, Fischer Cat. # S271-10, make up 5M solution.

l. Tween-20, Fischer Cat. # S337-500.

m. Na₃VO₄, Fischer Cat. # S454-50, to 1.8 g material add 80 ml MilliQH₂O, adjust pH to 10.0 with HCl or NaOH, boil in microwave, cool, checkpH, repeat procedure until pH stable at 10.0, add MilliQ H₂O to 100 mltotal volume, make 1 ml aliquots and store at −80° C.

n. MgCl₂, Fischer Cat. # M33-500, make up 1M solution.

o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ H₂O, add 59.6 gmaterial, adjust pH to 7.5, bring volume to 250 ml total, sterilefilter.

p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30 grams material addsterile distilled water to make total volume of 300 ml, store at 40° C.

q. TBST Buffer: to approx. 900 ml dH₂O in a 1 L graduated cylinder add6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to 7.2 withHCl, add 1.0 ml Triton X-100 and bring to 1 L total volume with dH₂O.

r. Goat Affinity purified antibody Rabbit IgG (whole molecule), CappelCat. # 55641.

s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz ChemicalCat. # SC-161.

t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada, etal., Oncogene, 8:2381-2390 (1993).

u. Sodium Carbonate Buffer, (Na₂CO₄, Fischer Cat. # S495):

to 10.6 g material add 800 ml MilliQ H₂O, when dissolved adjust pH to9.6 with NaOH, bring up to 1 L total volume with MilliQ H₂O, filter,store at 4° C.

2. Procedure

All of the following steps are conducted at room temperature unless itis specifically indicated otherwise. All ELISA plate washing is byrinsing 4× with TBST.

A. EMR Lysis

This procedure can be performed the night before or immediately prior tothe start of receptor capture.

1. Quick thaw lysates in a 37° C. waterbath with a swirling motion untilthe last crystals disappear.

2. Lyse cell pellet with 1×HNTG containing 1 mM PMSF. Use 3 ml of HNTGper 15 cm dish of cells. Add ½ the calculated HNTG volume, vortex thetube for 1 min., add the remaining amount of HNTG, vortex for anothermin.

3. Balance tubes, centrifuge at 10,000×g for 10 min at 4° C.

4. Pool supernatants, remove an aliquot for protein determination.

5. Quick freeze pooled sample in dry ice/ethanol bath. This step isperformed regardless of whether lysate will be stored overnight or usedimmediately following protein determination.

6. Perform protein determination using standard bicinchoninic acid (BCA)method (BCA Assay Reagent Kit from Pierce Chemical Cat. # 23225).

B. ELISA Procedure

1. Coat Corning 96 well ELISA plates with 5 μg per well Goat anti-Rabbitantibody in Carbonate Buffer for a total well volume of 50 μl. Storeovernight at 4° C.

2. Remove unbound Goat anti-rabbit antibody by inverting plate to removeliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash 4×with TBST. Pat plate on a paper towel to remove excess liquidand bubbles.

5. Add 1 μg per well of Rabbit anti-Met antibody diluted in TBST for atotal well volume of 100 μl.

6. Dilute lysate in HNTG (90 μg lysate/100 μl)

7. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

8. Wash 4×with TBST. Pat on paper towel to remove excess liquid andbubbles.

9. Add 50 μl of 1×lysate buffer per well.

10. Dilute compounds/extracts 1:10 in 1×Kinase Buffer in a polypropylene96 well plate.

11. Transfer 5.5 μl of diluted drug to ELISA plate wells. Incubate atroom temperature with shaking for 20 min.

12. Add 5.5 μl of 60 μM ATP solution per well. Negative controls do notreceive any ATP. Incubate at room temperature for 90 min., with shaking.

13. Wash 4× with TBST. Pat plate on paper towel to remove excess liquidand bubbles.

14. Add 100 μl per well of RC20 (1:3000 dilution in Blocking Buffer).Incubate 30 min. at room temperature with shaking.

15. Wash 4× with TBST. Pat plate on paper towel to remove excess liquidand bubbles.

16. Add 100 μl per well of Turbo-TMB. Incubate with shaking for 30-60min.

17. Add 100 μl per well of 1M H₂SO₄ to stop reaction.

18. Read assay on Dynatech MR7000 ELISA reader.

Test Filter=450 nm, reference filter=410 nm.

g. Biochemical arc assay—ELISA

This assay is used to determine src protein kinase activity measuringphosphorylation of a biotinylated peptide as the readout.

Materials and Reagents:

a. Yeast transformed with src from Courtneidge Laboratory (Sugen, Inc.,Redwood City, Calif.).

b. Cell lysates: Yeast cells expressing src are pelleted, washed oncewith water, re-pelleted and stored at −80° C. until use.

c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standardprocedures well known to those skilled in the art.

d. DMSO: Sigma, St. Louis, Mo.

e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flat BottomPlate, Corning Cat. #25805-96.

f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:Applied Scientific Cat. # A-72092.

g. Vecastain ELITE ABC reagent: Vector, Burlingame, Calif.

h. Anti-src (327) mab: Schizosaccharomyces Pombe is used to expressrecombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634;Superti-Furga, et al., Nature Biochem., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade210) is grown as described andtransformations are pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to repress expression from the nmtl promoter or in theabsence of thiamine to induce expression.

i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be usedinstead).

j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical.

2. Buffer Solutions:

a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. #450-1300EB.

b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS.

c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. # S495, make up 100 mMstock solution.

d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml (from a1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml(from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml totalvolume with MilliQ H₂O.

e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 ml total volumewith MilliQ H₂O.

f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml)

g. TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H₂O add 121.14 gmaterial, adjust pH to 7.5 with HCl, bring to 1 L total volume withMilliQ H₂O.

h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQH₂O.

i. Na₃VO₄: Fischer Cat. # S454-50; to 80 ml MilliQ H₂O, add 1.8 gmaterial; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool;check pH, repeat pH adjustment until pH remains stable afterheating/cooling cycle; bring to 100 ml total volume with MilliQ H₂O;make 1 ml aliquots and store at −80° C.

j. MgCl₂: Fischer Cat. # M33-500, make up 1M stock solution with MilliQH₂O.

k. HEPES: Fischer Cat. # BP 310-500; too 200 ml MilliQ H2O, add 59.6 gmaterial, adjust pH to 7.5, bring to 250 ml total volume with MilliQH₂O, sterile filter (1M stock solution).

l. TBST Buffer: TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and 8.766 gNaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 Ltotal volume with dH₂O.

m. MnCl₂: Fischer Cat. # M87-100, make up 1M stock solution with MilliQH₂O.

n. DTT: Fischer Cat. # BP172-5.

o. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 g TRIS and8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mlKinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQH₂O.

q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stock solution (1mM, 2.98 mg/ml) in water fresh just before use.

r. Vectastain ELITE ABC reagent: To prepare 14 ml of working reagent,add 1 drop of reagent A to 15 ml TBST and invert tube several times tomix. Then add 1 drop of reagent B. Put tube on orbital shaker at roomtemperature and mix for 30 minutes.

3. Procedures:

a. Preparation of src coated ELISA plate.

1. Coat ELISA plate with 0.5 μg/well anti-src mab in 100 μl of pH 9.6sodium carbonate buffer at 4° C. overnight.

2. Wash wells once with PBS.

3. Block plate with 0.15 ml 5% milk in PBS for 30 min. at roomtemperature.

4. Wash plate 5× with PBS.

5. Add 10 μg/well of src transformed yeast lysates diluted in LysisBuffer (0.1 ml total volume per well). (Amount of lysate may varybetween batches.) Shake plate for 20 minutes at room temperature.

b. Preparation of phosphotyrosine antibody-coated ELISA plate.

1. 4G10 plate: coat 0.5 μg/well 4G10 in 100 μl PBS overnight at 4° C.and block with 150 μl of 5% milk in PBS for 30 minutes at roomtemperature.

C. Kinase assay procedure.

1. Remove unbound proteins from step 1-7, above, and wash plates 5×withPBS.

2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of10×Kinase Buffer and 10 μM (final concentration)biotin-EEEYEEYEEEYEEEYEEEY per well diluted in water.

3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

4. Start kinase reaction by adding 10 μl/well of 0.05 mM ATP in water (5μM ATP final).

5. Shake ELISA plate for 15 min. at room temperature.

6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate fromsection B, above.

8. Incubate for 30 min. while shaking at room temperature.

9. Wash plate 5× with TBST.

10. Incubate with Vectastain ELITE ABC reagent (100 μl/well) for 30 min.at room temperature.

11. Wash the wells 5× with TBST.

12. Develop with Turbo TMB.

h. Biochemical lck Assay—ELISA

This assay is used to determine lck protein kinase activities measuringphosphorylation of GST-ζ as the readout.

1. Materials and Reagents:

a. Yeast transformed with lck. Schizosaccharomyces Pombe is used toexpress recombinant Lck (Superti-Furga, et al., EMBO J, 12:2625-2634;Superti-Furga, et al., Nature Biotech., 14:600-605). S. Pombe strainSP200 (h-s leul.32 ura4 ade210) is grown as described andtransformations with pRSP expression plasmids are done by the lithiumacetate method (Superti-Furga, supra). Cells are grown in the presenceof 1 μM thiamine to induce expression.

b. Cell lysates: Yeast cells expressing lck are pelleted, washed once inwater, re-pelleted and stored frozen at −80° C. until use.

c. GST-ζ: DNA encoding for GST-ζ fusion protein for expression inbacteria obtained from Arthur Weiss of the Howard Hughes MedicalInstitute at the University of California, San Francisco. Transformedbacteria are grown overnight while shaking at 25° C. GST-ζ is purifiedby glutathione affinity chromatography, Pharmacia, Alameda, Calif.

d. DMSO: Sigma, St. Louis, Mo.

e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat BottomPlate, Corning Cat. #25805-96.

f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:Applied Scientific Cat. # AS-72092.

g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia)Cat. #90001605.

h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301.

i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. # 5215-005-003.

j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat # sc-433.

k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may be usedinstead).

2. Buffer solutions:

a. PBS (Dulbecco's Phosphate-Buffered Saline) 1X solution: GIBCO PBS,GIBCO Cat. # 450-1300EB.

b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS-pH7.5, 58.44 g NaCl, 10 mlTween-20, bring up to 1 L total volume with MilliQ H₂O.

c. Carbonate Buffer: Na₂CO₄ from Fischer, Cat. # S495;

make up 100 mM solution with MilliQ H₂O.

d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl₂; 0.2 ml (from a1M stock solution) MnCl₂; 0.2 ml (from a 1M stock solution) DTT; 5.0 ml(from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml totalvolume with MilliQ H₂O.

e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from5M stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a100 mM stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1ml Na₃VO₄ (from a 0.1 M stock solution); bring to 100 ml total volumewith MilliQ H₂O.

f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml).

g. TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H₂O add 121.14 gmaterial, adjust pH to 7.5 with HCl, bring to 1 L total volume withMilliQ H₂O.

h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQH₂O.

i. Na₃VO₄: Fischer Cat. # S454-50; to 80 ml MilliQ H₂O, add 1.8 gmaterial; adjust pH to 10.0 with HCl or NaOH; boil in a microwave; cool;check pH, repeat pH adjustment until pH remains stable afterheating/cooling cycle; bring to 100 ml total volume with MilliQ H₂O;make 1 ml aliquots and store at −80° C.

j. MgCl₂: Fischer Cat. # M33-500, make up 1M stock solution with MilliQH₂O.

k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ H₂O, add 59.6 gmaterial, adjust pH to 7.5, bring to 250 ml total volume with MilliQH₂O, sterile filter (1M stock solution).

l. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150 ml MilliQ H₂O add30 g material, bring 300 ml total volume with MilliQ H₂O, filter through0.22 μm filter, store at 4° C.

m. TBST Buffer: To 900 ml dH₂O add 6.057 g TRIS and 8.766 g NaCl; adjustpH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total volumewith dH₂O.

n. MnCl₂: Fischer Cat. # M87-100, make up 1M stock solution with MilliQH₂O.

o. DTT; Fischer Cat. # BP172-5.

p. TBS (TRIS Buffered Saline): to 900 ml MilliQ H₂O add 6.057 g TRIS and8.777 g NaCl; bring to 1 L total volume with MilliQ H₂O.

q. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 mlKinase Buffer, 200 μg GST-ζ, bring to final volume of 8.0 ml with MilliQH₂O.

2. Procedures:

a. Preparation of Lck coated ELISA plate.

1. Coat 2.0 μg/well Sheep anti-mouse IgG in 100 μl of pH 9.6 sodiumcarbonate buffer at 4° C. overnight.

2. Wash well once with PBS.

3. Block plate with 0.15 ml of blocking Buffer for 30 min. at room temp.

4. Wash plate 5×with PBS.

5. Add 0.5 μg/well of anti-lck (mab 3A5) in 0.1 ml PBS at roomtemperature for 1-2 hours.

6. Wash plate 5×with PBS.

7. Add 20 μg/well of lck transformed yeast lysates diluted in LysisBuffer (0.1 ml total volume per well). (Amount of lysate may varybetween batches) Shake plate at 4° C. overnight to prevent loss ofactivity.

b. Preparation of phosphotyrosine antibody-coated ELISA plate.

1. UB40 plate: 1.0 μg/well UB40 in 100 μl of PBS overnight at 4° C. andblock with 150 μl of Blocking Buffer for at least 1 hour.

c. Kinase assay procedure.

1. Remove unbound proteins from step 1-7, above, and wash plates 5× withPBS.

2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 μl of10×Kinase Buffer and 2 μg GST-ζ per well diluted with water).

3. Add 10 μl of compound diluted in water containing 10% DMSO andpre-incubate for 15 minutes at room temperature.

4. Start kinase reaction by adding 10 μl/well of 0.1 mM ATP in water (10μM ATP final).

5. Shake ELISA plate for 60 min. at room temperature.

6. Stop kinase reaction by adding 10 μl of 0.5 M EDTA per well.

7. Transfer 90 μl supernatant to a blocked 4G10 coated ELISA plate fromsection B, above.

8. Incubate while shaking for 30 min. at room temperature.

9. Wash plate 5× with TBST.

10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in 100 μlTBST for 30 min. at room temperature.

11. Wash the wells SX with TBST.

12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100μl of TBST for 30 min. at room temperature.

13. Wash the wells 5× with TBST.

14. Develop with Turbo TMB.

i. Assay Measuring Phosphorylating Function of RAF

The following assay reports the amount of RAF-catalyzed phosphorylationof its target protein MEK as well as MEK's target MAPK. The RAF genesequence is described in Bonner et al., 1985, Molec. Cell. Biol. 5:1400-1407, and is readily accessible in multiple gene sequence databanks. Construction of the nucleic acid vector and cell lines utilizedfor this portion of the invention are fully described in Morrison etal., 1988, Proc. Natl. Acad. Sci. USA 85: 8855-8859.

Materials and Reagents

1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, Md.

2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mMPMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;

3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression andpurification by affinity chromatography are performed according to themanufacturer's procedures. Catalog# K 350-01 and R 350-40, InvitrogenCorp., San Diego, Calif.

4. His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cellstransformed with pUC18 vector encoding His-MAPK. His-MAPK is purified byNi-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, Calif.,as described herein.

5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa. Catalog,# 515-006-008, Lot# 28563

6. RAF-1 protein kinase specific antibody: URP2653 from UBI.

7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL,Gaithersburg, Md.

8. Wash buffer: TBST-50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% TritonX-100

9. Block buffer: TBST, 0.1% ethanolamine pH 7.4

10. DMSO, Sigma, St. Louis, Mo.

11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1% TritonX-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium ortho vanadate, 0.5 MMDTT and 10 mM MgCl₂.

12. ATP mix: 100 mM MgCl₂, 300 mM ATP, 10 mCi 33P ATP (Dupont-NEN)/mL.

13 Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa.

14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland.

15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, Pa.

16. Tomtec plate harvester, Wallac, Turku, Finnland.

17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland.

18. NUNC 96-well V bottom polypropylene plates for compounds AppliedScientific Catalog # AS-72092.

Procedure

All of the following steps are conducted at room temperature unlessspecifically indicated.

1. ELISA plate coating: ELISA wells are coated with 100 ml of Sheep antimouse affinity purified antiserum (1 mg/100 mL coating buffer) overnight at 4° C. ELISA plates can be used for two weeks when stored at 4°C.

2. Invert the plate and remove liquid. Add 100 mL of blocking solutionand incubate for 30 min.

3. Remove blocking solution and wash four times with wash buffer. Patthe plate on a paper towel to remove excess liquid.

4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for1 hour. Wash as described in step 3.

5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to10 mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for1 hour. Shake the plate during incubation. Negative controls receive nolysate. Lysates from RAS/RAF infected Sf9 insect cells are preparedafter cells are infected with recombinant baculoviruses at a MOI of 5for each virus, and harvested 48 hours later. The cells are washed oncewith PBS and lysed in RIPA buffer. Insoluble material is removed bycentrifugation.(5 min at 10 000×g). Aliquots of lysates are frozen indry ice/ethanol and stored at −80° C. until use.

6. Remove non-bound material and wash as outlined above (step 3).

7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust thevolume to 40 mL with kinase buffer. Methods for purifying T-MEK and MAPKfrom cell extracts are provided herein by example.

8. Pre-dilute compounds (stock solution 10 mg/mL DMSO) or extracts 20fold in TBST plus 1% DMSO. Add 5 mL of the pre-dilutedcompounds/extracts to the wells described in step 6. Incubate for 20min. Controls receive no drug.

9. Start the kinase reaction by addition of 5 mL ATP mix; Shake theplates on an ELISA plate shaker during incubation.

10. Stop the kinase reaction after 60 min by addition of 30 mL stopsolution to each well.

11. Place the phosphocellulose mat and the ELISA plate in the Tomtecplate harvester. Harvest and wash the filter with the filter washsolution according to the manufacturers recommendation. Dry the filtermats. Seal the filter mats and place them in the holder. Insert theholder into radioactive detection apparatus and quantify the radioactivephosphorous on the filter mats.

Alternatively, 40 mL aliquots from individual wells of the assay platecan be transferred to the corresponding positions on thephosphocellulose filter mat. After air drying the filters, put thefilters in a tray. Gently rock the tray, changing the wash solution at15 min intervals for 1 hour. Air-dry the filter mats. Seal the filtermats and place them in a holder suitable for measuring the radioactivephosphorous in the samples. Insert the holder into a detection deviceand quantify the radioactive phosphorous on the filter mats.

j. CDK2/Cyclin A—Inhibition Assay

This assay analyzes the protein kinase activity of CDK2 in exogenoussubstrate.

Reagents

A. Buffer A (80 mM Tris (pH 7.2), 40 mM MgCl₂): 4.84 G. Tris (F.W.=121.1g/mol), 4.07 g. MgCl₂ (F.W.=203.31 g/mol) dissolved in 500 ml H₂O.Adjust pH to 7.2 with HCl.

B. Histone H1 solution (0.45 mg/ml Histone H1 and 20 mM HEPES pH 7.2 (pH7.4 is OK): 5 mg Histone H1 (Boehinger Mannheim) in 11.111 ml 20 mMHEPES pH 7.2 (477 mg HEPES (F.W.=238.3 g/mol) dissolved in 100 ml ddH₂O,stored in 1 ml aliquots at −80° C.

C. ATP solution (60 μM ATP, 300 μg/ml BSA, 3 mM DTT): 120 μl 10 mM ATP,600 μl 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at −80° C.

D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, 0.5mM DTT, 10% glycerol, stored in 9 μl aliquots at −80° C.

Description of Assay

1. Prepare solutions of inhibitors at three times the desired finalassay concentration in ddH₂O/15% DMSO by volume.

2. Dispense 20 μl of inhibitors to wells of polypropylene 96-well plates(or 20 μl 15% DMSO for positive and negative controls).

3. Thaw Histone H1 solution (1 ml/plate), ATP solution (1 ml/plate plus1 aliquot for negative control), and CDK2 solution (9 μl/plate). KeepCDK2 on ice until use. Aliquot CDK2 solution appropriately to avoidrepeated freeze-thaw cycles.

4. Dilute 9 μl CDK2 solution into 2.1 ml Buffer A (per plate). Mix.Dispense 20 μl into each well.

5. Mix 1 ml Histone H1 solution with 1 ml ATP solution (per plate) intoa 10 ml screw cap tube. Add γ³³P ATP to a concentration of 0.15 μCi/20μl (0.15 μCi/well in assay). Mix carefully to avoid BSA frothing. Add 20μl to appropriate wells. Mix plates on plate shaker. For negativecontrol, mix ATP solution with an equal amount of 20 mM HEPES pH 7.2 andadd γ³³P ATP to a concentration of 0.15 μCi/20 μl solution. Add 20 μl toappropriate wells.

6. Let reactions proceed for 60 minutes.

7. Add 35 μl 10% TCA to each well. Mix plates on plate shaker.

8. Spot 40 μl of each sample onto P30 filter mat squares. Allow mats todry (approx. 10-20 minutes).

9 Wash filter mats 4×10 minutes with 250 ml 1% phosphoric acid (10 mlphosphoric acid per liter ddH₂O).

10. Count filter mats with beta plate reader.

2. Cellular/Biologic Assays

Assay 1: PDGF-Induced BrdU Incorporation Assay Materials and Reagents

(1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat.

No. 1 647 229, Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen, Inc.,Redwood City, Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human PDGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (PDGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 2: EGF-Induced BrdU Incorporation Assay Materials and Reagents

(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen, Inc.,Redwood City, Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human EGF-R.

Protocol

(1) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, ina 96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (EGF) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 3: EGF-Induced Her2-Driven BrdU Incorporation Materials andReagents

(1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4, made in house.

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line engineered to express a chimeric receptor having theextra-cellular domain of EGF-R and the intra-cellular domain of Her2.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) on day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and testcompounds are added to the cells simultaneously. The negative controlwells receive serum free DMEM with 0.1% BSA only; the positive controlcells receive the ligand (EGF) but no test compound. Test compounds areprepared in serum free DMEM with ligand in a 96 well plate, and seriallydiluted for 7 test concentrations.

(4) After 20 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 01% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

Assay 4: IGF1-Induced BrdU Incorporation Assay Materials and Reagents

(1) IGF1 Ligand: human, recombinant; G511, Promega Corp,. USA.

(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229,Boehringer Mannheim, Germany.

(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use,Cat. No. 1 647 229, Boehringer Mannheim, Germany.

(6) PBS Washing Solution: 1X PBS, pH 7.4, made in house (Sugen, Inc.,Redwood City, Calif.).

(7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma ChemicalCo., USA.

(8) 3T3 cell line genetically engineered to express human IGF-1receptor.

Protocol

(1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a96-well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

(2) After 24 hours, the cells are washed with PBS, and then are serumstarved in serum free medium (0%CS DMEM with 0.1% BSA) for 24 hours.

(3) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) andtest compounds are added to the cells simultaneously. The negativecontrol wells receive serum free DMEM with 0.1% BSA only; the positivecontrol cells receive the ligand (IGF1) but no test compound. Testcompounds are prepared in serum free DMEM with ligand in a 96 wellplate, and serially diluted for 7 test concentrations.

(4) After 16 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration=10 μM) for 1.5 hours.

(5) After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

(6) The FixDenat solution is thoroughly removed by decanting and tappingthe inverted plate on a paper towel. Milk is added (5% dehydrated milkin PBS, 200 μl/well) as a blocking solution and the plate is incubatedfor 30 minutes at room temperature on a plate shaker.

(7) The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

(8) The antibody conjugate is thoroughly removed by decanting andrinsing the wells 5 times with PBS, and the plate is dried by invertingand tapping on a paper towel.

(9) TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

(10) The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

g. HUV-EC-C Assay

The following protocol may also be used to measure a compound's activityagainst PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which arenaturally expressed by HUV-EC cells.

DAY 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection; catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS; obtained from GibcoBRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% try sin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company; catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco;catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific; catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200 g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL; catalogue no. 21127-014)+0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter, Coulter Electronics, Inc.) and add assay medium to the cells toobtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well; incubate ˜24 h at 37° C., 5% CO₂.

DAY 1

1. Make up two-fold drug titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of drugat 200 μM (4×the final well concentration) to the top well of aparticular plate column. Since the stock drug concentration is usually20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

Therefore, diluent made up to 2% DMSO in assay medium (F12K+0.5% fetalbovine serum) is used as diluent for the drug titrations in order todilute the drug but keep the DMSO concentration constant. Add thisdiluent to the remaining wells in the column at 60 μl/well. Take 60 μlfrom the 120 μl of 200 μM drug dilution in the top well of the columnand mix with the 60 μl in the second well of the column. Take 60 μl fromthis well and mix with the 60 μl in the third well of the column, and soon until two-fold titrations are completed. When the next-to-the-lastwell is mixed, take 60 μl of the 120 μl in this well and discard it.Leave the last well with 60 μl of DMSO/media diluent as anon-drug-containing control. Make 9 columns of titrated drug, enough fortriplicate wells each for 1) VEGF (obtained from Pepro Tech Inc.,catalogue no. 100-200, 2) endothelial cell growth factor (ECGF) (alsoknown as acidic fibroblast growth factor, or aFGF) (obtained fromBoehringer Mannheim Biochemica, catalogue no. 1439 600); or, 3) humanPDGF B/B (1276-956, Boehringer Mannheim, Germany) and assay mediacontrol. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the drug dilutions to the 96-well assay platescontaining the 0.8-1.0×10⁴ cells/100 μl/well of the HUV-EC-C cells fromday 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each drug condition. As with the drugs, the growthfactor concentrations are 4× the desired final concentration. Use theassay media from day 0 step 2 to make the concentrations of growthfactors. Incubate approximately 24 hours at 37° C., 5% CO₂. Each wellwill have 50 μl drug dilution, 50 μl growth factor or media, and 100 ulcells,=200 ul/well total. Thus the 4× concentrations of drugs and growthfactors become 1×once everything has been added to the wells.

DAY 2

1. Add ³H-thymidine (Amersham; catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

DAY 3

1. Freeze plates overnight at −20° C.

DAY 4

1. Thaw plates and harvest with a 96-well plate harvester (TomtecHarvester 96®) onto filter mats (Wallac; catalogue no. 1205-401); readcounts on a Wallac Betaplate™ liquid scintillation counter.

3. In Vivo Animal Models

A. Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastrointestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619),A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung,ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cellsand NIH 3T3 fibroblasts genetically engineered to overexpress EGFR,PDGFR, IGF-1R or any other test kinase. The following protocol can beused to perform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450×g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

B. Tumor Invasion Model

The following tumor invasion model has been developed and maybe used forthe evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases, to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurements of tumor size, grade of invasion,immunochemistry, and in situ hybridization).

D. Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index: IC₅₀/LD₅₀, IC₅₀, the dose required toachieve 50% inhibition, can be measured using standard techniques suchas those described herein. LD₅₀ the dosage which results in 50%toxicity, can also be measured by standard techniques (Mossman, 1983, J.Immunol. Methods, 65:55-63), by measuring the amount of LDH released(Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64:313; Deckerand Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by measuringthe lethal dose in animal models. Compounds with a large therapeuticindex are preferred. The therapeutic index should be greater than 2,preferably at least 10, more preferably at least 50.

Conclusion

Thus, it will be appreciated that the compounds of the present inventionare expected to show improved hydrosolubility and the compounds, methodsand pharmacological compositions of the present invention are expectedto modulate PTK activity and therefore to be effective as therapeuticagents against PTK-related disorders.

Although certain embodiments and examples have been used to describe thepresent invention, it will be apparent to those skilled in the art thatchanges to the embodiments and examples shown may be made withoutdeparting from the scope and spirit of the invention.

Other embodiments are within the following claims.

What is claimed:
 1. A 3-heteroarylidenyl-2-azaindolinone compound havingthe following chemical structure:

wherein, A is selected from the group consisting of nitrogen, oxygen andsulfur and it is understood that when A is oxygen or sulfur, R³ does notexits; only one of B, D, E, F and G is nitrogen and it is understoodthat when B, D, E, F or G is nitrogen, R⁴, R⁵, R⁶ or R⁷, respectively,do not exist; Z is selected from the group consisting of oxygen, sulfurand NR¹¹ wherein, R¹¹ is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, C-carboxy, C-amido,guanyl, sulfonyl and trihalomethanesulfonyl; R¹ is selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,hydroxy, alkoxy, C-carboxy, C-amido, trihalomethanecarbonyl,trihalomethane-sulfonyl and sulfonyl; R² is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl and halogen; when A isnitrogen, R³ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, hydroxy, alkoxy, aryloxy, carbonyl, C-carboxy,O-carboxy, C-amido, guanyl, sulfonyl and trihalomethanesulfonyl; one ortwo of R⁴, R⁵, R⁶ and R⁷ are independently selected from the groupconsisting of —NR⁸R⁹, —J(CH₂)_(m)NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and—NHR¹⁰; wherein, J is selected from the group consisting of oxygen, NHand sulfur; m is 0, 1, 2 or 3; Y is selected from the group consistingof NH and oxygen; Q is selected from the group consisting of hydroxy,alkoxy, aryloxy, amino, N-hydroxylamino, O-carboxy, NR⁸R⁹ andN-peptidyl; R⁸ and R⁹ are independently selected from the groupconsisting of hydrogen, alkyl, C-carboxy, C-peptidyl and, combined, afive-member or 6-member heteroalicyclic group containing at least onenitrogen; R¹⁰ is a polyhydroxyalkyl group; those of R⁴, R⁵, R⁶ and R⁷which are not substituted as noted above are independently selected fromthe group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl,S-sulfonamido, N-sulfonamido, trihalomethanesulfonyl, carbonyl,C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo,O-thiocarbamyl, N-thiocarbamyl, guanyl and phosphonyl; R⁴ and R⁵ maycombine to form a six-member cycloalkyl, heteroaryl or heteroalicyclicring; and the physiologically acceptable salt thereof.
 2. The compoundor salt of claim 1 wherein R¹ is selected from the group consisting ofhydrogen and alkyl.
 3. The compound or salt of claim 2 wherein Z isoxygen.
 4. The compound or salt of claim 3 wherein R² is hydrogen. 5.The compound or salt of claim 4 wherein A is sulfur.
 6. The compound orsalt of claim 5 wherein R⁴ and R⁵, combined, form a six-membercycloalkyl ring.
 7. The compound or salt of claim 6 wherein R⁶ isselected from the group consisting of —NR⁸R⁹, —J(CH₂)_(m)NR⁸R⁹,—J(CH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and —NHR¹⁰.
 8. The compound or salt ofclaim 7 wherein R⁷ is selected from the group consisting of —NR⁸R⁹,—J(CH₂)_(m)NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and —NHR¹⁰.
 9. Thecompound or salt of claim 4 wherein A is nitrogen.
 10. The compound orsalt of claim 9 wherein R³ is hydrogen.
 11. The compound or salt ofclaim 10 wherein R⁴ and R⁵ are lower alkyl.
 12. The compound or salt ofclaim 11 wherein R⁶ is selected from the group consisting —NR⁸R⁹,—J(CH₂)_(m)NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and —NHR¹⁰.
 13. Thecompound or salt claim 12 wherein R⁷ is selected from the groupconsisting of —NR⁸R⁹, —J(CH₂)_(m)NR⁸R⁹, —J(CH₂)_(m)C(═Y)Q, —N═CNR⁸R⁹ and—NHR¹⁰.
 14. The compound or salt of claim 4 wherein A and B arenitrogen.
 15. The compound or salt of claim 4 wherein A is oxygen.
 16. Apharmacological composition of said compound or salt of claim
 1. 17. Amethod for treating a protein tyrosine kinase related disorder in anmammal comprising administering a therapeutically effective amount ofsaid pharmacological composition of claim 16 to said mammal and whereinsaid protein tyrosine kinase related disorder is blastoglioma, Kaposi'ssarcoma, melanoma, lung cancer, ovarian cancer or prostate cancer.
 18. Amethod for treating a protein tyrosine kinase related disorder in anmammal comprising administering a therapeutically effective amount ofsaid pharmacological composition of claim 16 to said mammal and whereinsaid protein tyrosine kinase related disorder is squamous cellcarcinoma, astrocytoma, glioblastoma, head cancer, neck cancer, lungcancer or bladder cancer.
 19. A method for treating a protein tyrosinekinase related disorder in an mammal comprising administering atherapeutically effective amount of said pharmacological composition ofclaim 16 to said mammal and wherein said protein tyrosine kinase relateddisorder related disorder is breast cancer, small-cell lung cancer, orgliomas.
 20. A method for treating a protein tyrosine kinase relateddisorder in an mammal comprising administering a therapeuticallyeffective amount of said pharmacological composition of claim 16 to saidmammal and wherein said protein tyrosine kinase related disorder iscolorectal cancer, thyroid cancer, pancreatic carcinoma, gastriccarcinoma, leukemia, lymphoma, Hodgkin's disease or Burkitts disease.21. A method for treating a protein tyrosine kinase related disorder inan mammal comprising administering a therapeutically effective amount ofsaid pharmacological composition of claim 16 to said mammal and whereinsaid protein tyrosine kinase related disorder is arthritis, diabeticretinopathy, hepatic cirrhosis, atherosclerosis, glomerulonephritis,diabetic neuropathy, thrombic microangiopathy syndromes, transplantrejection, or diabetes.
 22. The method according to any one of claims17, 18, 19, 20 or 21 wherein said mammal is a human.